Anti-HLA-DR antibody

ABSTRACT

This invention provides an anti-HLA-DR monoclonal antibody. This invention relates to an antibody binding to HLA-DR or a functional fragment thereof having (a) life-extending effects in nonhuman animals bearing HLA-DR-expressing cancer cells and (b) activity of suppressing immune responses lower than that of L243, or an antibody binding to HLA-DR or a functional fragment thereof exhibiting immunosuppressive activity equivalent to or higher than that of the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55).

TECHNICAL FIELD

The present invention relates to an anti-HLA-DR antibody that recognizesthe human leucocyte antigen-DR (HLA-DR), which is a cell membranemolecule associated with immunity. Further, the present inventionrelates to the following two types of agents comprising, as an activeingredient, the anti-HLA-DR antibody: (1) a preventive or therapeuticagent for diseases caused by an HLA-DR-expressing cell, especially, atherapeutic agent for malignant tumors; and (2) a preventive ortherapeutic agent for immune responses caused by an HLA-DR-expressingcell, especially, a therapeutic agent for chronic rheumatism.

BACKGROUND ART

The use of an antibody, which binds to a protein expressed on a cellsurface and is capable of leading the cell to death or toxicity, hasbeen attempted in the treatment of cancer, etc. At present, a chimericantibody (Rituximab) targeting CD20, which is a receptor existing on acell membrane, and a monoclonal antibody such as a humanized antibodytargeting Her2/neu are used in the treatment of malignant tumors, andtheir therapeutic effects are acknowledged. An antibody is characterizedby a long serum half-life and high specificity for an antigen, and thusis particularly useful as an anti-tumor agent. For example, when anantibody targeting a tumor-specific antigen is administered,accumulation thereof in the tumor is presumed. Thus, an attack by theimmune system due to the complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC) on cancer cells can beexpected. Binding of a radionuclide or an agent such as a cytotoxicsubstance to the antibody enables the effective transmission of thebound agent to a tumor site. This also reduces the amount of the agentreaching other non-specific tissues, and thus reduced side effects canbe expected. When a tumor-specific antigen has activity for inducingcell death, an agonistic antibody is administered. In contrast, when atumor-specific antigen is associated with growth and survival of cells,a neutralizing antibody is administered. This can result in theaccumulation of tumor-specific antibodies and arrest or regression oftumor growth due to the activity of the antibody. As mentioned above,antibodies are considered suitable for application as anti-tumor agentsbecause of their features.

Recently, significant anti-tumor effects of Rituximab have beenexhibited with respect to B-cell lymphoma, and the side effects thereofare limited. Since Rituximab is a chimeric human-mouse protein, however,the antigenicity of Rituximab itself is strong, an antibody against amouse moiety is produced inside the body, and the effect could bedeteriorated. For some types of cancer, the therapeutic effect ofRituximab is low with the use of Rituximab alone, and the combined usethereof with an anticancer agent is currently being clinically examined(see McLaughlin P. et. al., J Clin Oncol. (1998), 16, 2825–2833;Coiffier B. et al., Blood (1998), 92, 1927–81). Accordingly, a novelanti-tumor antibody targeting an antigen is needed, and a monoclonalantibody against HLA-DR, which is a class II major histocompatibilitycomplex (MHC) molecule, can be expected to have clinical anti-tumoractivity as an antibody that recognizes an antigen different from thatrecognized by Rituximab.

In contrast, class II major histocompatibility complex (MHC) moleculesbind to antigen peptide fragments and present these antigen peptidefragments to helper (CD4⁺) T-cells (“Th” cells) (see Babbin B. et al.,Nature (1985), 317, 359–361). A monoclonal antibody that is specific forthe class II MHC molecules is reported as a very potent selectiveinhibitor against the immune response of Th cells in vitro (seeBaxevanis C N, et. al., Immunogenetics (1980), 11, 617–625). Since thismonoclonal antibody was discovered, it has been considered to be anagent that can be used in the selective immunosuppressive therapy ofautoimmune diseases such as chronic rheumatism. Based on the initial invivo research, significant effects of these monoclonal antibodies on Thcellular heterogeneity and autoimmune response have been elucidated (seeRosenbaum J T. et al., J. Exp. Med. (1981), 154, 1694–1702; Waldor M K.et al., Proc. Natl. Acad. Sci. USA (1983), 80, 2713–2717; Jonker M. etal., J. Autoimmun. (1988), 1, 399–414; Stevens H P. et al., Transplant.Proc. (1990), 22, 1783–1784). Further, as a result of research usingprimates, it was discovered that graft-versus-host disease in homograftwas suppressed (Billing R. & Chatterjee S. (1983), Transplant. Proc.,15, 649–650; Jonker M. et al., Transplant Proc. (1991), 23, 264–265).

Currently, immunological rejection at the time of organ transplantationis clinically suppressed using immunosuppressive agents such ascyclosporin A or FK506. A disadvantage of these immunosuppressive agentsis that potent side effects are caused by the non-specific suppressionof the immune response.

Accordingly, antibodies are considered suitable for use asimmunosuppressive agents with few side effect because of their features.

DISCLOSURE OF THE INVENTION

An immunosuppressive agent using a human antibody having highimmunosuppressive activity and low immunogenicity has not yet beendeveloped.

An object of the present invention is to produce such an antibody anduse it as an anti-tumor agent or immunosuppressive agent.

The present inventors have conducted concentrated studies in order toproduce an antibody against human HLA-DR. As a result, they havesucceeded in obtaining a monoclonal antibody exhibiting an anti-tumoreffect at very low concentration on HLA-DR-expressing cancer cells and amonoclonal antibody that specifically suppresses immune activity throughHLA-DR. Further, they have identified the sequence in the variableregion of the monoclonal antibody and determined the epitope to whichthe monoclonal antibody binds. This has led to the completion of thepresent invention.

More specifically, the present invention is as follows.

The present invention provides, in the first aspect thereof, amonoclonal antibody that binds to HLA-DR produced from a mouse-mousehybridoma, for example, a monoclonal antibody that is preferably a humanantibody produced from HD4, HD6, HD8, or HD10, or a functional fragmentthereof. The monoclonal antibody produced from HD4, HD6, HD8, or HD10 isof an immunoglobulin G (IgG) type. The hybridoma HD8 and the hybridomaHD10 are deposited internationally at the International Patent OrganismDepositary of the National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)as of Oct. 11, 2001 under the accession numbers FERM BP-7773 and FERMBP-7774, respectively. The hybridoma HD4 is deposited internationally atthe International Patent Organism Depositary of the National Instituteof Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Oct. 11, 2001 under theaccession number FERM BP-7771. The hybridoma HD6 is depositedinternationally at the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of Oct.11, 2001 under the accession number FERM BP-7772.

According to an embodiment of the present invention, the antibodyaccording to the present invention comprises a variable region of theantibody produced from the aforementioned hybridoma or a functionalfragment thereof.

In another embodiment of the present invention, the antibody accordingto the present invention includes an antibody having a modifiedsubclass, which is an antibody produced from the hybridoma HD8 having anIgG1, IgG2, IgG3, or IgG4 subclass or a functional fragment thereof, anantibody produced from the hybridoma HD4 having an IgG1, IgG2, IgG3, orIgG4 subclass or a functional fragment thereof, an antibody producedfrom the hybridoma HD10 having an IgG1, IgG2, IgG3, or IgG4 subclass ora functional fragment thereof, or an antibody produced from thehybridoma HD6 having an IgG1, IgG2, IgG3, or IgG4 subclass or afunctional fragment thereof. According to a further embodiment of thepresent invention, the antibody according to the present invention is anantibody having a modified amino acid sequence in the constant region ofthe heavy chain or a functional fragment thereof. For example, anantibody comprises amino acid 331 in the constant region of the heavychain according to the EU numbering system (see Sequences of Proteins ofImmunological Interest, NIH Publication No. 91-3242) being substitutedwith Ser or a functional fragment thereof.

In another embodiment of the present invention, the subclass of theantibody or a functional fragment thereof is rearranged to IgG1, IgG2,or IgG4, amino acid 331 in the constant region of the heavy chainaccording to the EU numbering system is substituted with Ser, and thesubclass is modified as IgG1, IgG1Ser, IgG2, IgG2Ser, or IgG4. As aresult, only an antibody or a functional fragment thereof having an IgG1or IgG1Ser subclass develops ADCC, and only an antibody or a functionalfragment thereof having an IgG1 or IgG2 subclass develops CDC activity.

In another aspect of the present invention, the present inventionprovides an antibody that binds to HLA-DR or a functional fragmentthereof comprising a variable region of an antibody produced from thehybridoma HD4, HD6, HD8, or HD10. In an embodiment of the presentinvention, the antibody or a functional fragment thereof according tothe present invention comprises a variable region of an antibodyproduced from the hybridoma HD8 having an amino acid sequence in themature variable region of the amino acid sequences as shown in SEQ IDNOs: 21 and 23. In another embodiment of the present invention, theantibody or a functional fragment thereof according to the presentinvention comprises a variable region of an antibody produced from thehybridoma HD4 having the amino acid sequence in the mature variableregions in the amino acid sequences as shown in SEQ ID NOs: 17 and 19.

In a further aspect of the present invention, the present inventionrelates to an antibody or a functional fragment thereof that can bind toa specific epitope of HLA-DR. In embodiments of the present invention,the antibody according to the present invention is an antibody thatbinds to HLA-DR or a functional fragment thereof, which maximally bindsto the peptide as shown in SEQ ID NO: 82. This peptide is selected fromamong peptides that are prepared by shifting 2 amino acids of the aminoacids in the extracellular region (the amino acid sequence being shownin SEQ ID NO: 147 and the nucleotide sequence being shown in SEQ ID NO:146) of the HLA-DR β chain (DRB1*15011) to prepare 13-mer peptides(preparing 13-mer peptides for 199 amino acids, i.e., amino acids 29 to227, in the amino acid sequence as shown in SEQ ID NO: 147), binding theresulting peptides to a cellulose membrane through the C-terminus, andacetylating the N-terminus. Also, the antibody according to the presentinvention is an antibody that binds to HLA-DR or a functional fragmentthereof, which potently binds to all three peptides as shown in SEQ IDNOs: 82, 83, and 84. These peptides are selected from among peptidesthat are prepared by shifting 2 amino acids of the amino acids in theextracellular region of the HLA-DR β chain (DRB1*15011) to prepare13-mer peptides, binding the resulting peptides to a cellulose membranethrough the C-terminus, and acetylating the N-terminus. Further, theantibody according to the present invention is an antibody that binds toHLA-DR or a functional fragment thereof, which significantly binds toall the peptides as shown in SEQ ID NOs: 24 to 39 and all the peptidesas shown in SEQ ID NOs: 40 to 43. These peptides are prepared byshifting 2 amino acids of the amino acids in the extracellular region ofthe HLA-DR β chain (DRB1*15011) to prepare 13-mer peptides, binding theresulting peptides to a cellulose membrane through the C-terminus, andacetylating the N-terminus.

Furthermore, the present invention provides, in another aspect, anantibody or a functional fragment thereof that can extend the survivalof individual mice by suppressing tumor growth (for example, thosederived from the Raji cell transplanted to SCID mice). The amount of theantibody or a functional fragment thereof according to the presentinvention to be administered to tumor-bearing test animals (for example,tumor-bearing test animals such as lymphoma cell-bearing mouse models,each with a body weight of 20 g) is 0.1 μg/body to 1 μg/body or 5 μg/kgto 50 μg/kg. Examples of dose are 1 μg/body or 50 μg/kg, and preferably0.1 μg/body or 5 μg/kg.

In an embodiment of the present invention, the antibody according to thepresent invention binds to HLA-DR or a functional fragment thereof,which has the following properties (a) and (b):

(a) when a 6-week-old SCID mouse is inoculated intravenously with 10 μlof the anti-asialo GM1 antiserum, on the next day, inoculatedintravenously with 5×10⁶ of Burkitt's lymphoma cells Raji (ATCC CCL-86),and 5 days thereafter, inoculated with 5 to 50 μg/kg, based on bodyweight, of the antibody of the present invention, the survival ratio ofthe mouse is higher than that achieved when inoculated with the sameamount of the human anti-HSA antibody; and

(b) the immunosuppressive activity is lower than that achieved whenusing the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55) at thesame concentration, wherein the immunosuppressive activity is assayed asfollows: 50 μl of antibody adjusted at 8 μg/mL, and preferably 8 μg/mLusing 10% FCS-containing RPMI 1640 medium is mixed with 50 μL of maturedendritic cell suspension derived from a first human donor adjusted at2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium in wells of a96-well plate, the mixture is allowed to stand at 4° C. for 30 minutes,the resultant is mixed with 100 μL of T-cell suspension (purity: 99% orhigher) adjusted at 1×10⁶ cells/mL using 10% FCS-containing RPMI 1640medium derived from a second human donor having a histocompatibleantigen different from that of the first human donor, the mixture iscultured at 37° C. in the presence of 5% CO₂ for 5 days, ³H thymidine isadded thereto at 1.0 μCi/well, the resultant is cultured at 37° C. inthe presence of 5% CO₂ for 16 to 20 hours, the ³H thymidine incorporatedin the cell is recovered and then measured using a scintillator, and theincorporation of the ³H thymidine into the cell is used as an indicatorto assay the immunosuppressive activity.

Further, the present invention provides, in another aspect thereof, anantibody recognizing HLA-DR or a functional fragment thereof, whichexhibits immunosuppressive activity equivalent to or higher than that ofthe mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55). In anembodiment of the present invention, the antibody or a functionalfragment thereof according to the present invention has theimmunosuppressive activity equivalent to or higher than that achievedwhen using the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)at the same concentration, wherein the immunosuppressive activity isassayed in the following manner. First, 50 μl of antibody adjusted at 8μg/mL using 10% FCS-containing RPMI 1640 medium is mixed with 50 μL ofmature dendritic cell suspension derived from a first human donoradjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium inwells of a 96-well plate. The mixture is then allowed to stand at 4° C.for 30 minutes, and the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1 x 106 cells/mL using10% FCS-containing RPMI 1640 medium derived from a second human donorhaving a histocompatible antigen different from that of the first humandonor. The resultant is cultured at 37° C. in the presence of 5% CO₂ for5 days, 3H thymidine is added thereto at 1.0 μCi/well, the resultant isfurther cultured at 37° C. in the presence of 5% CO2 for 16 to 20 hours,the ³H thymidine incorporated in the cell is recovered and then measuredusing a scintillator, and incorporation of the ³H thymidine into thecell is used as an indicator to assay the immunosuppressive activity.

The present invention further provides, in another aspect, a nucleicacid encoding an antibody comprising a variable region of an antibodyproduced from a hybridoma or a functional fragment thereof, wherein saidnucleic acid is possessed by a hybridoma selected from the groupconsisting of the hybridoma HD8 (accession number FERM BP-7773), thehybridoma HD10 (accession number FERM BP-7774), the hybridoma HD4(accession number FERM BP-7771), and the hybridoma HD6 (accession numberFERM BP-7772), a protein encoded by the nucleic acid, an expressionvector having the nucleic acid, and a host selected from the groupconsisting of E. coli, yeast cell, insect cell, mammalian cell, plantcell, and mammalians having the expression vector. The present inventionprovides in its embodiments: a nucleic acid encoding the antibody or afunctional fragment thereof, which comprises a variable region having anamino acid sequence of the mature variable regions of the amino acidsequences as shown in SEQ ID NOs: 17 and 19; a nucleic acid encoding theantibody or a functional fragment thereof, which comprises a variableregion having an amino acid sequence of the mature variable regions ofthe amino acid sequences as shown in SEQ ID NOs: 21 and 23; and anucleic acid encoding the antibody or a functional fragment thereof,wherein the antibody is selected from the group consisting of theantibody HD8G1Ser, the antibody HD8G2Ser, and the antibody HD4G2Ser. Theantibody HD8G1Ser is the antibody HD8 having an IgG1 subclass and aminoacid 331 according to the EU numbering system being substituted withSer, the antibody HD8G2Ser is the antibody HD8 having an IgG2 subclassand amino acid 331 according to the EU numbering system beingsubstituted with Ser, and the antibody HD4G2Ser is the antibody HD4having an IgG2 subclass and amino acid 331 according to the EU numberingsystem being substituted with Ser.

The present invention further provides, in another aspect thereof, aprocess for producing the anti-HLA-DR monoclonal antibody, wherein agene encoding the anti-HLA-DR monoclonal antibody is isolated from ahybridoma selected from the group consisting of the hybridoma HD8(accession number FERM BP-7773), the hybridoma HD10 (accession numberFERM BP-7774), the hybridoma HD4 (accession number FERM BP-7771), andthe hybridoma HD6 (accession number FERM BP-7772), an expression vectorhaving said gene is constructed, the expression vector is introducedinto a host to express the monoclonal antibody, and the anti-HLA-DRmonoclonal antibody is collected from the resulting host, a culturesupernatant of the host, or a secretion product of the host.

The present invention further provides, in another aspect thereof, apreventive, therapeutic, or diagnostic agent for tumors, whichcomprises, as an active ingredient, the aforementioned antibody or afunctional fragment thereof.

Examples of tumors that can be prevented or treated include at least onemember selected from the group consisting of leukemia (including chroniclymphatic leukemia and acute lymphatic leukemia), lymphoma (includingnon-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-celllymphoma, Burkitt's lymphoma, malignant lymphoma, diffuse lymphoma, andfollicular lymphoma), myeloma (including multiple myeloma), breastcancer, colon cancer, kidney cancer, gastric cancer, ovarian cancer,pancreatic cancer, cervical cancer, endometrial cancer, esophagealcancer, liver cancer, head and neck squamous cancer, skin cancer,urinary tract cancer, prostate cancer, choriocarcinoma, pharyngealcancer, laryngeal cancer, pleural tumor, arrhenoblastoma, endometrialhyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma,angioma, cavernous angioma, hemangioblastoma, retinoblastoma,spongiocytoma, neurofibroma, oligodendroglioma, medulloblastoma,neuroblastoma, neuroglioma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor.

The present invention provides, in another aspect, an immunosuppressiveagent comprising, as an active ingredient, the antibody or a functionalfragment thereof according to the present invention. The presentinvention further provides a preventive, therapeutic, or diagnosticagent for autoimmune diseases or allergies, which comprises, as anactive ingredient, the antibody or a functional fragment thereofaccording to the present invention.

In an embodiment of the present invention, a preventive or therapeuticagent is an immunosuppressive agent at the time of organ transplantation(a preventive or therapeutic agent for immunological rejection at thetime of pancreatic islet or kidney transplantation or GVHD), atherapeutic agent for autoimmune diseases (for example, rheumatism,arteriosclerosis, multiple sclerosis, systemic erythematodes, idiopathicthrombocythemia, or Crohn's disease), or a preventive or therapeuticagent for allergic diseases such as asthma. In another embodiment of thepresent invention, the life-extending effects are recognized in thetumor-bearing SCID mice to which the Raji cell had been transplanted 5days after the tumor transplantation with the administration of 5 μg/kgor lower of the antibody or a functional fragment thereof according tothe present invention.

The present invention includes an antibody or a functional fragmentcomprising the amino acid sequences in the mature variable region of theheavy chain and that of the light chain of the antibody produced fromthe hybridoma HD4 as shown in SEQ ID NO: 17 or 19 and the amino acidsequences in the mature variable region of the heavy chain and that ofthe light chain of the antibody produced from the hybridoma HD8 as shownin SEQ ID NO: 21 or 23.

The aforementioned antibody or a functional fragment thereof comprises,for example, the amino acid sequence in the mature variable region ofthe heavy chain and that of the light chain encoded by the nucleic acidsequence isolated from the hybridoma HD4 as shown in SEQ ID NO: 16 or 18and the amino acid sequence in the mature variable region of the heavychain and that of the light chain encoded by the nucleic acid sequenceisolated from the hybridoma HD8 as shown in SEQ ID NO: 20 or 22.

The present invention is hereafter described in detail.

It is also reported that the anti-HLA-DR monoclonal antibody hasactivity of suppressing immune responses. Based on the initial in vivoresearch, significant effects of the anti-HLA-DR monoclonal antibody onTh cellular heterogeneity and autoimmune response were elucidated (seeRosenbaum J T. et al., J. Exp. Med. (1981), 154, 1694–1702; Waldor M K.et al., Proc. Natl. Acad. Sci. USA (1983), 80, 2713–2717; Jonker M. etal., J. Autoimmun. (1988), 1, 399–414; Stevens H P. et al., Transplant.Proc. (1990), 22, 1783–1784). Further, as a result of research usingprimates, it was discovered that graft-versus-host disease in homograftwas suppressed (Billing R. & Chatterjee S. (1983), Transplant. Proc.,15, 649–650; Jonker M. et al., Transplant Proc. (1991), 23, 264–265).These reported antibodies, however, are mouse antibodies. Recently,Protein Design Labs Inc. has developed a humanized HLA-DR antibody usingthe mouse anti-HLA-DR antibody 1D10 and converting regions other thanthe variable region into the sequence of the human antibody by theirhumanizing techniques and gene recombination (see Sheri A K. et. al.,Int. J. Cancer (2001), 93, 556–565). This is clinically examined in theU.S.

The novel human anti-HLA-DR monoclonal antibody according to the presentinvention is a complete human antibody, and the antigenicity against themouse sequences, which is always problematic in the mouse antibody, hasalready been resolved.

Any of the immunoglobulin G (IgG), immunoglobulin A (IgA),immunoglobulin E (IgE), and immunoglobulin M (IgM) antibodies can besuitably used. In general, IgG is preferable.

The terms used in the present invention are defined in order to describethe present invention in more detail.

1. HLA-DR and an Antibody thereof

The antibody according to the present invention is an antibody againstHLA-DR, which is a class II major histocompatibility complex (MHC),i.e., an antibody that recognizes and binds to HLA-DR and an antibodythat has reactivity with HLA-DR.

The “antibody that binds to HLA-DR” in the present invention is anantibody or a portion thereof having reactivity with human HLA-DR or aportion thereof, and it includes a functional fragment thereof. A“functional fragment” refers to a portion of an antibody (a partialfragment), which has at least one function of the antibody on anantigen. Specific examples thereof include F(ab′)₂, Fab′, Fab, Fv,disulphide-linked Fv, single-chain Fv (scFv), and a polymer thereof (D.J. King, Applications and Engineering of Monoclonal Antibodies, 1998, T.J. International Ltd.). Or, a “functional fragment” is a fragment of anantibody that is capable of binding to an antigen. A functional fragmentof the antibody according to the present invention binds to HLA-DR andexhibits an anti-tumor effect or potent immunosuppressive activity.

The term “human antibody” used herein refers to an antibody, which is anexpression product of a human-derived antibody gene. The human antibodycan be obtained by introducing a human antibody locus and administeringan antigen to a transgenic animal that is capable of producing ahuman-derived antibody as described below. An example of such atransgenic animal is a mouse, and a process for producing a mouse thatcan produce a human antibody is described in WO 02/43478.

Examples of the antibody according to the present invention includevarious antibodies exhibiting anti-tumor effects at low concentration onhuman HLA-DR-expressing cancer cells as described in the examples below.

The antibody according to the present invention includes a monoclonalantibody comprising the heavy chain and/or light chain having an aminoacid sequence with deletion, substitution, or addition of one or severalamino acids in various amino acid sequences thereof. The aforementionedpartial modification of amino acid (deletion, substitution, insertion,or addition) can be introduced into the amino acid sequence of theantibody according to the present invention by partially modifying thenucleotide sequence encoding the amino acid sequence of interest. Suchpartial modification of the nucleotide sequence can be introduced by ageneral method of conventional site specific mutagenesis (Proc Natl AcadSci USA, 1984, Vol. 81: 5662). The antibody used herein refers to animmunoglobulin in which all regions including a variable region and aconstant region of the heavy chain and a variable region and a constantregion of the light chain constituting the immunoglobulin are derivedfrom a gene encoding the immunoglobulin.

The antibody according to the present invention includes an antibodyhaving any immunoglobulin class and isotype.

The anti-HLA-DR antibody according to the present invention can beproduced by a process as described below. Specifically, human HLA-DR, apart thereof, a binding product thereof with a suitable carriersubstance for enhancing antigenicity of the antigen (e.g., bovine serumalbumin), or the like is administered to a non-human mammalian, such asa human antibody-producing transgenic mouse, for immunization togetherwith an immunopotentiating agent (e.g., Freund's complete or incompleteadjuvant), if necessary. Alternatively, a gene encoding the human HLA-DRα chain or β chain can be introduced, and an animal cell havingexcessively expressed HLA-DR on its surface can be administered forimmunization. A monoclonal antibody can be obtained by culturing ahybridoma obtained by fusing an antibody-producing cell obtained fromthe immunized animal and a myeloma cell incapable of producing anautoantibody, and selecting a clone that produces a monoclonal antibodyhaving specific affinity to the antigen used for the immunization.

The antibody according to the present invention includes those havingdifferent subclasses that were modified by genetic engineering known toa person skilled in the art (see, for example, EP 314161). Specifically,an antibody having a subclass that is different from the originalsubclass can be obtained using DNA encoding a variable region of theantibody according to the present invention using a genetic engineeringtechnique. For example, the subclass of the antibody according to thepresent invention can be converted into IgG2 or IgG4 to obtain anantibody having a low degree of binding to the Fc receptor. On thecontrary, the subclass of the antibody according to the presentinvention can be converted into IgG1 or IgG3 to obtain an antibodyhaving a high degree of binding to the Fc receptor. Further,modification of the amino acid sequence in the constant region of theantibody according to the present invention by genetic engineering orbinding with a sequence of a constant region having such a sequenceenables changing of the degree of binding to the Fc receptor (seeJaneway C A. Jr. and Travers P. (1997), Immunobiology, Third Edition,Current Biology Ltd./Garland Publishing Inc.) or that to the complement(see Mi-Hua Tao, et al., 1993, J. Exp. Med). The antibody according tothe present invention includes these antibodies having modified aminoacid sequences in the constant regions. Modification of the amino acidsequence refers to deletion, substitution, or addition of one or severalamino acids of the amino acid sequence. For example, the sequence CCCencoding proline (P) 331 in the constant region of the heavy chainaccording to the EU numbering system (see Sequences of proteins ofimmunological interest, NIH Publication No. 91-3242) is varied to TCCencoding serine (S) to substitute proline with serine, thereby changingthe degree of binding to the complement. In the case of an anticanceragent, when the antibody itself does not have activity of inducing celldeath, a preferable antibody has anti-tumor activity due to theantibody-dependent cellular cytotoxicity (ADCC) or complement-dependentcytotoxicity (CDC) through the Fc receptor. When the antibody itself hasactivity of inducing cell death, an antibody having a low degree ofbinding to the Fc receptor may be preferable. In the case of animmunosuppressive agent, an antibody having no ACDD or CDC activity ispreferable when, for example, only the binding between the T-cell andthe antigen-presenting cell is three-dimensionally suppressed. When ADCCor CDC activity could cause toxicity, an antibody having resolved thetoxicity-causing activity by varying the Fc portion or changing thesubclass may be preferable.

The antibody according to the present invention includes a modifiedantibody, the subclass of which has been rearranged to IgG1, IgG2, orIgG4, and a modified antibody, in which amino acid 331 in the constantregion of the heavy chain of the modified antibody, the subclass ofwhich has been rearranged to IgG1 or IgG2, according to the EU numberingsystem, has been substituted with Ser to constitute IgG1Ser or IgG2Ser.Specifically, the present invention includes an antibody in which onlyIgG1 and the IgG1Ser exhibit ADCC activity and only IgG1 and the IgG2exhibit CDC activity as a result of the subclass modification into IgG1,IgG1Ser, IgG2, IgG2Ser, or IgG4.

Therapeutic effects on diseases such as cancer can be further enhancedby binding, for example, radionuclides such as iodine, yttrium, indium,and technetium (J. W. Goding, Monoclonal Antibodies: principles andpractice, 1993, ACADEMIC PRESS), bacterial toxins such as Pseudomonasexotoxin, diphtheria toxin, and ricin, chemotherapeutants such asmethotrexate, mitomycin, and calicheamicin (D. J. King, Applications andEngineering of Monoclonal Antibodies, 1998, T. J. International Ltd, M.L. Grossbard, Monoclonal Antibody-Based Therapy of Cancer, 1998, MarcelDekker Inc), or a prodrug such as maytansinoid (Chari et al., CancerRes., 1992, Vol. 52: 127, Liu et al., Proc Natl. Acad Sci USA, 1996,Vol. 93: 8681) to the antibody according to the present invention.

In the present invention, the following steps are included in theproduction of a monoclonal antibody: (1) purification of a biopolymerthat is used as an immunogen and/or production of a cell havingexcessively expressed antigen proteins on its surface; (2) production ofan antibody-producing cell by immunizing an animal by injecting anantigen, sampling blood to assay the antibody titer, and determining thestage of excising the spleen or the like; (3) preparation of myelomacells; (4) cell fusion between an antibody-producing cell and themyeloma cell; (5) selection of a group of hybridomas producing anantibody of interest; (6) division (cloning) into a single cell clone;(7) if necessary, culture of a hybridoma for mass-producing monoclonalantibodies or breeding animals to which the hybtridoma has beentransplanted; (8) examination of physiological activity or recognitionspecificity of the thus produced monoclonal antibody or examination ofproperties thereof as labeling reagents; and the like.

A process for producing the anti-HLA-DR monoclonal antibody is hereafterdescribed in detail along with the above steps, although the process forproducing the antibody is not limited thereto. For example, anantibody-producing cell other than a splenic or myeloma cell can also beused.

(1) Purification of Antigen

A transformant is obtained by incorporating DNA encoding the HLA-DR αchain and β chain into an expression vector for animal cells andintroducing the expression vector into an animal cell. This can be usedas an antigen in that state. Since the primary structures of the HLA-DRα chain and β chain are known (Steven G E, et al., (2000), The HLAFactsBook, Academic Press), a peptide is chemically synthesized from theamino acid sequence of HLA-DR by a method known to a person skilled inthe art, and the resultant can be used as an antigen.

A cell in which the full-length α chain and β chain of the human HLA-DRare introduced into the L929 cell and HLA-DR heterodimers areexcessively expressed on its surface is also effective as an immunogen.pEF-neo-HLA-DRa and pEF-neo-HLA-DRβ can be produced by separatelyincorporating DNA encoding the human HLA-DR α chain protein and DNAencoding the human HLA-DR β chain protein into the expression vector foranimal cells, pEF-neo. pEF-neo is a vector comprising aneomycin-resistant gene incorporated into modified pEF-BOS (seeMizushima S. & Nagata S., Nucleic Acids Res (1990), 18, 5332). It shouldbe noted that DNA encoding HLA-DR, a vector, a host, or the like is notlimited thereto.

Specifically, a transformant obtained by transforming the L929 cell inpEF-neo-HLA-DRα and pEF-neo-HLA-DRβ is cultured, and confirmation ofHLA-DR expression using the trait of neomycin resistance acquired by thecell to which the pEF-neo vector has been inserted and the goatanti-HLA-DR polyclonal antibody (DAKO) are employed as indicators,thereby producing the L929 cell having excessively expressed humanHLA-DR on its surface.

(2) Step of Preparing Antibody-producing Cell

The antigen obtained in (1) is mixed with an adjuvant such as Freund'scomplete or incomplete adjuvant or potash alum, and experimental animalsare immunized with the resultant as the immunogen. A transgenic mousecapable of producing a human-derived antibody is most suitably used asan experimental animal, and such a mouse is described in literature byTomizuka et al. (Tomizuka et al., Proc Natl Acad Sci USA, 2000, Vol. 97:722).

An immunogen can be administered at the time of mouse immunization byany of hypodermic injection, intraperitoneal injection, intravenousinjection, endodermic injection, intramuscular injection, or plantarinjection. Intraperitoneal injection, plantar injection, or intravenousinjection is preferable.

Immunization can be performed once or several times at suitableintervals (preferably at intervals of 2 to 4 weeks). Thereafter, theantibody titer against the antigen in the serum of the immunized animalis assayed, and the animal having a sufficiently high antibody titer isused as a source of antibody-producing cells. This can enhance theeffects of the subsequent procedure. In general, the antibody-producingcell derived from an animal 3 to 5 days after the final immunization canbe preferably used for the later cell fusion.

Examples of a method for measuring the antibody titer that is usedherein include various conventional techniques such as radioimmunoassay(hereafter referred to as “RIA”), enzyme-linked immunosorbent assay(hereafter referred to as “ELISA”), fluorescent antibody technique, andpassive haemagglutination. From the viewpoints of detection sensitivity,rapidity, accuracy, the possibility of automating operations, and thelike, the use of RIA or ELISA is preferable.

In the present invention, the antibody titer can be assayed in thefollowing manner in accordance with, for example, ELISA. At the outset,an antibody against a human antibody is allowed to adsorb on the surfaceof the solid phase such as a 96-well plate for ELISA. Subsequently, thesurface of the solid phase having no antigen adsorbed thereon is coveredwith a protein that is unrelated to an antigen, for example, bovineserum albumin (BSA), the surface is washed, it is brought into contactwith a gradually-diluted sample (for example, mouse serum) as a primerantibody, and the anti-HLA-DR antibody in the sample is then bound tothe antigen. Further, an antibody against an enzyme-labeled humanantibody is added as a secondary antibody and bound to a human antibody,followed by washing. Thereafter, the substrate of the enzyme is added,and changes of the absorbance, etc. are assayed based on the colorationdue to substrate decomposition. Thus, the antibody titer is calculated.

(3) Step of Preparing Myeloma Cell

A cell incapable of producing autoantibodies derived from a mammaliansuch as a mouse, rat, guinea pig, hamster, rabbit, or human can be usedas a myeloma cell. In general, established cell lines obtained from amouse, for example, 8-azaguanine-resistant mouse (BALB/c-derived)myeloma cells P3X63Ag8U.1 (P3-U1) (Yelton, D. E. et al., Current Topicsin Microbiology and Immunology, 81, 1–7 (1978)), P3/NSI/1-Ag4-1 (NS-1)(Kohler, G. et al. European J. Immunology, 6 511–519 (1976)), Sp2/O-Ag14(SP-2) (Shulman, M. et al. Nature, 276, 269–270 (1978)), P3X63Ag8.653(653) (Kearney, J. F. et al. J. Immunology, 123, 1548–1550 (1979)), andP3X63(X63) (Horibata, K. and Harris, A. W. Nature, 256, 495–497 (1975))are preferably used. These cell lines are subjected to subculture insuitable medium, for example, 8-azaguanine medium (medium prepared byadding 8-azaguanine to RPMI-1640 medium comprising glutamine,2-mercaptoethanol, gentamicin, and fetal calf serum (hereafter referredto as “FCS”), Iscove's Modified Dulbecco's Medium (hereafter referred toas “IMDM”), or Dulbecco's Modified Eagle Medium (hereafter referred toas “DMEM”). These cell lines are subjected to subculture in normalmedium (for example, DMEM containing 10% FCS) 3 to 4 days before thecell fusion to reliably have 2×10⁷ or more cells on the day of the cellfusion.

(4) Cell Fusion

An antibody-producing cell is a plasma cell or a lymphocyte as itsprecursor cell. This may be obtained from any site of an individual andcan be generally obtained from, for example, the spleen, lymph node,bone marrow, tonsilla, peripheral blood, or suitable combinationsthereof, with the spleen cell being most commonly used.

After the final immunization, a site containing an antibody-producingcell therein, for example, the spleen, is excised from a mouse having apredetermined antibody titer to produce a spleen cell, which is anantibody-producing cell. The means for fusing this spleen cell with themyeloma cell obtained in step (3) that is most commonly performed atpresent is a method using polyethylene glycol having relatively lowcytotoxicity and simple fusion operations. This method comprises, forexample, the following procedures.

Spleen cells and myeloma cells are thoroughly washed in serum-freemedium (for example, DMEM) or phosphate-buffered saline (hereafterreferred to as “PBS”) and mixed with each other to bring the ratio ofspleen cells to myeloma cells to approximately 5:1 to 10:1, followed bycentrifugation. The supernatant is removed, the precipitated group ofcells is thoroughly unraveled, and 1 mL of serum-free medium containing50% (w/v) polyethylene glycol (molecular weight: 1,000 to 4,000) is thenadded thereto dropwise while stirring. Thereafter, 10 mL of serum-freemedium is slowly added thereto, followed by centrifugation. Thesupernatant is discarded again, the precipitated cells are suspended ina suitable amount of normal medium containing ahypoxanthine-aminopterin-thymidine (hereafter referred to as “HAT”)solution and human interleukin 6 (hereafter referred to as “IL-6”) (thismedium is hereafter referred to as “HAT medium”), the resultant isfractionated in each well of the culture plate (hereafter referred to asa “plate”), and cultured in the presence of 5% CO₂ at 37° C. forapproximately 2 weeks. During the culture, HAT medium is suitablysupplemented.

(5) Selection of a Group of Hybridomas

When the aforementioned myeloma cells are 8-azaguanine-resistant, i.e.,when they are hypoxanthine-guanine phosphoribosyltransferase (HGPRT)deficient, non-fused myeloma cells and cells fused between myeloma cellscannot survive in HAT-containing medium. While cells fused betweenantibody-producing cells or hybridomas of antibody-producing cells andmyeloma cells can survive, the survival time of cells fused betweenantibody-producing cells is limited. Accordingly, continuation ofculture in HAT-containing medium results in survival of only hybridomasof antibody-producing cells and myeloma cells. In consequence, thisenables the selection of hybridomas.

HAT medium for the hybridomas grown as a colony is replaced with amedium from which aminopterin has been removed (hereafter referred to as“HT medium”). Thereafter, a part of the culture supernatant is collectedto assay the anti-HLA-DR antibody titer by, for example, ELISA. When theaforementioned fusion protein is used as an antigen for ELISA, anoperation of eliminating a clone is required so as not to select a clonethat produces an antibody which specifically binds to the Fc region ofthe human IgG. The presence or absence of such a clone can be inspectedby, for example, ELISA using the Fc region of the human IgG as anantigen.

A process using a 8-azaguanine-resistant cell line was exemplifiedabove, although other cell lines can be also used depending on theprocess used for selecting a hybridoma. In such a case, the compositionof the medium to be used is also changed.

(6) Step of Cloning

The antibody titer is assayed in the same manner as described in (2),and the hybridomas, which were found to produce specific antibodies, aretransferred to the other plate to perform cloning. Examples of cloningprocesses include: limiting dilution in which culture is conducted bydiluting, so that one hybridoma is contained in a well of the plate; thesoft agar method in which culture is conducted in a soft agar medium andcolonies are recovered; a method in which culture is conducted byremoving one cell using a micromanipulator, and a “sorter clone”process, in which one cell is separated using a cell sorter. Limitingdilution is simple and often employed.

The wells, the antibody titers of which have been recognized, arerepeatedly subjected to cloning by, for example, limiting dilution 2 to4 times, and those having stable antibody titers are selected asanti-HLA-DR monoclonal antibody-producing hybridomas.

The mouse-mouse hybridomas HD8, HD10, HD4, and HD6, the humananti-HLA-DR monoclonal antibody-producing cells according to the presentinvention, are deposited internationally at the International PatentOrganism Depositary of the National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan) as of Oct. 11, 2001. The accession number of thehybridoma HD8 is FERM BP-7773, that of the hybridoma HD10 is FERMBP-7774, that of the hybridoma HD4 is FERM BP-7771, and that of thehybridoma HD6 is FERM BP-7772.

(7) Preparation of Monoclonal Antibody by Culturing Hybridomas

The hybridomas, which have completed the cloning process, are culturedin normal medium, which is a replacement of the HT medium. Large-scaleculture is carried out by rotation culture in a large culture flask,spinner culture, or culture in a hollow-fiber system. The supernatantobtained through this large-scale culture is purified using a techniqueknown to a person skilled in the art such as gel filtration. Thisenables the production of the anti-HLA-DR monoclonal antibody thatcomprises, as an active ingredient, the preventive or therapeutic agentof the present invention. Also, the hybridoma is multiplied in theabdominal cavity of a mouse of the same lineage (e.g., BALB/c), a nu/numouse, rat, guinea pig, hamster, or rabbit. This can provide ascitesfluid containing a large amount of the anti-HLA-DR monoclonal antibodiescomprising, as an active ingredient, the preventive or therapeutic agentof the present invention. In order to simply carry out the purification,a commercially available monoclonal antibody purification kit (e.g.,MabTrap GII Kit, Amersham Pharmacia Biotech) or the like can be used.

The thus obtained monoclonal antibody has high antigen specificity forthe human HLA-DR.

(8) Examination of Monoclonal Antibody

The isotype and the subclass of the thus obtained monoclonal antibodycan be identified in the following manner. Examples of a method foridentification include the Ouchterlony method, ELISA, and RIA. Althoughthe Ouchterlony method is simple, this method requires a concentratingoperation when the concentration of the monoclonal antibody is low. Inthe case of ELISA or RIA, however, the isotype and the subclass of themonoclonal antibody can be identified by allowing the culturesupernatant to react with the antigen-adsorbed solid phase, and usingantibodies, as secondary antibodies, that correspond to variousimmunoglobulin isotypes and subclasses.

Proteins can be quantified by the Folin-Lowry method or by calculationbased on the absorbance at 280 nm (1.4 (OD 280)=immunoglobulin 1 mg/ml).

The epitope recognized by the monoclonal antibody can be identified inthe following manner. Various partial constructs of molecules recognizedby the monoclonal antibody are first prepared. Partial constructs can beprepared by, for example, a method in which various partial peptides ofthe molecule are prepared using a conventional technique of synthesizingoligopeptides or a method in which a DNA sequence encoding a partialpeptide of interest is incorporated in a suitable expression plasmidusing a gene recombination technique to produce partial constructsinside or outside a host such as E. coli. These methods are generallyused in combinations to attain the above object. For example, severalpolypeptide sequences, the lengths of which were successively andsuitably shortened from the C-terminus or N-terminus of the antigenprotein, are prepared by a gene recombination technique known to aperson skilled in the art, and the reactivity of the monoclonal antibodytherewith is then examined to roughly determine the recognition site.

Thereafter, various oligopeptides in the corresponding site, variants ofthe peptides, or the like are synthesized using a technique ofsynthesizing oligopeptides known to a person skilled in the art. Theaffinity of the monoclonal antibody, which is an active ingredient ofthe preventive or therapeutic agent of the present invention, with thesepeptides is then inspected or competition suppressing activity ofpeptides against the binding between the monoclonal antibody and theantigen is inspected, thereby more precisely limiting the epitope. Inorder to simply obtain various oligopeptides, a commercially availablekit (for example, SPOTs Kit (Genosys Biotechnologies, Inc), a series ofMultipin Peptide Synthesis Kits using the multipin synthesis technique(Chiron Corporation), or the like) can be used.

Alternatively, a gene encoding a human monoclonal antibody is clonedfrom an antibody-producing cell such as a hybridoma, the clone productis incorporated into a suitable vector, and the resultant is introducedinto a host (e.g., a mammalian cell strain, E. coli, yeast cell, insectcell, or plant cell) to produce a recombinant antibody using a generecombination technique (P. J. Delves, ANTIBODY PRODUCTION ESSENTIALTECHNIQUES, 1997, WILEY, P. Shepherd and C. Dean, Monoclonal Antibodies,2000 OXFORD UNIVERSITY PRESS, J. W. Goding, Monoclonal Antibodies:principles and practice, 1993, ACADEMIC PRESS).

The present invention includes a nucleic acid comprising a gene sequenceof an antibody possessed by a hybridoma which produces the antibody ofthe present invention. More particularly, the present invention includesa nucleic acid that corresponds to the mature variable regions of theheavy chain and the light chain of the antibody produced from thehybridoma of the present invention as described below. The nucleic acidincludes DNA and RNA. The present invention includes a nucleic acid, thesequence of which is modified by means of substitution, deletion, and/oraddition of at least one nucleotide in the frame portion in the variableregion of the heavy or light chain (see FR1, FR2, FR3, and FR4:Sequences of proteins of immunological interest, NIH Publication No.91-3242), which hybridizes with a nucleic acid complementary to anucleic acid before sequence modification under stringent conditions,binds to HLA-DR, has (a) life-extending effects in HLA-DR-expressedcancer cell-bearing non-human animals and (b) lower activity ofsuppressing immune responses compared with that of L243, and encodes anantibody exhibiting immunosuppressive activity equivalent to or higherthan that of the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)in the nucleic acid comprising a gene sequence of the antibody possessedby a hybridoma which produces the antibody of the present invention. Theantibody refers to an immunoglobulin in which a variable region and aconstant region of the heavy chain and all regions including a variableregion and a constant region of the light chain constituting theimmunoglobulin are derived from a gene encoding the immunoglobulin.Stringent conditions involve the occurrence of hybridization only whenthe sequence is at least 90%, preferably at least 95%, and morepreferably at least 97% homologous with the DNA sequence encoding theantibody of the present invention. In general, these conditions involvethe occurrence of hybridization at a temperature about 5° C. to about30° C., and preferably about 10° C. to about 25° C. lower than themelting temperature of the perfect hybrid. Stringent conditions aredescribed in J. Sambrook et al., Molecular Cloning, A LaboratoryMannual, Second Edition, Cold Spring Harbor Laboratory Press (1989), andthe conditions described therein can be used.

In order to prepare a gene encoding the monoclonal antibody from ahybridoma, DNAs encoding the V region of the L chain, the C region ofthe L chain, the V region of the H chain, and the C region of the Hchain of the monoclonal antibody are prepared by PCR and the like. OligoDNA constructed from the anti-HLA-DR antibody gene or the amino acidsequence can be used as a primer, and DNA prepared from a hybridoma canbe used as a template. These DNAs are incorporated into a suitablevector, and the resultant is introduced into a host to be expressed.Alternatively, these DNAs are separately incorporated into each suitablevector for co-expression.

A phage or plasmid that can autonomously multiply in a hostmicroorganism is used as a vector. Examples of plasmid DNA include aplasmid derived from E. coli, Bacillus subtilis, or yeast, and anexample of phage DNA is λ phage.

A host that is used in transformation is not particularly limited aslong as the gene of interest can be expressed therein. Examples thereofinclude bacteria (e.g., E. coli or Bacillus subtilis), yeast, animalcells (e.g., COS cells or CHO cells), and insect cells.

A method for introducing a gene into a host is known, and examplesthereof include any methods such as a method using calcium ions,electroporation, spheroplast, the lithium acetate method, the calciumphosphate method, and lipofection. Examples of a method for introducinga gene into animals as described below include microinjection, a methodfor introducing a gene into the ES cell by electroporation orlipofection, and nucleus transplantation.

In the present invention, the anti-HLA-DR antibody can be obtained bycollecting it from a culture product obtained by culturing atansformant. The “culture product” refers to any of: (a) a culturesupernatant; (b) a cultured cell, cultured bacterial cell, or fragmentedproduct thereof; or (c) a secretion product of the transformant. Whenculturing a transformant, a medium that is suitable for the host ofinterest is used, and a culture method such as stationary culture orroller bottle culture is employed.

After the culture, when the protein of interest is produced in abacterial or other cell, the bacterial or other cell is destroyed tocollect the antibody. When the antibody of interest is produced outsidethe bacterial or other cell, the culture solution remaining unchanged isused, or the bacterial or other cell is removed by centrifugation, etc.Thereafter, general biochemical techniques using various types ofchromatography for isolation and purification of proteins are performedsingly or in suitable combinations. Thus, the antibody of interest canbe isolated and purified from the culture product.

With the use of a technique for preparing transgenic animals, animalhosts comprising the genes of the antibody of interest are incorporatedin endogenous genes. For example, transgenic cattle, goats, sheep, orpigs are prepared. From the milk secreted from these transgenic animals,a large amount of monoclonal antibodies derived from the antibody genesthereof can be obtained (Wright, G. et al., (1991), Bio/Technology 9,830–834). When culturing a hybridoma in vitro, a hybridoma ismultiplied, maintained, and stored in accordance with various conditionssuch as properties of the cells to be cultured, purposes of tests andresearch, or culture methods. A known nutrient medium that is used forproducing a monoclonal antibody in a culture supernatant or variousnutrient media derived and prepared from a known basal medium can beused to perform the culture.

(9) Properties of the Antibody

The antibody according to the present invention has the followingfunctional properties a) and b), and these properties can be confirmedby, for example, methods described for each item:

a) HLA-DR-expressing human cancer cells are transplanted inimmunodeficient mice such as SCID mice, the survival ratio of mice wheninoculated with the antibody of the present invention is inspected, andas a result, the number of days for which the mice survive is prolonged;and

b) activity of suppressing immune responses by allogeneic mixedlymphocyte reaction is lower than that of L243. More specifically, theproperties a) and b) are as follows:

(a) when a 6-week-old SCID mouse is inoculated intravenously with 10 μlof the anti-asialo GM1 antiserum, on the next day, inoculatedintravenously with 5×10⁶ of Burkitt's lymphoma cells Raji (ATCC CCL-86),and 5 days thereafter, inoculated once intravenously with 5 to 50 μg/kg,and preferably 5 μg/kg, based on body weight, of the antibody of thepresent invention, the survival ratio of the mouse 90 days after theinoculation is higher than that 90 days after the inoculation with thesame amount of the human anti-HSA antibody; and

(b) the immunosuppressive activity is lower than that achieved whenusing the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55) at thesame concentration, wherein the immunosuppressive activity is assayed asfollows: 50 μl of antibody adjusted at 8 to 200 μg/mL, and preferably 8μg/mL using 10% FCS-containing RPMI 1640 medium is mixed with 50 μL ofmature dendritic cell suspension derived from a first human donoradjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium inwells of a 96-well plate, the mixture is allowed to stand at 4° C. for30 minutes, the resultant is mixed with 100 μL of T-cell suspension(purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor,the mixture is cultured at 37° C. in the presence of 5% CO₂ for 5 days,³H thymidine is added thereto at 1.0 μCi/well, the resultant is culturedat 37° C. in the presence of 5% CO₂ for 16 to 20 hours, the ³H thymidineincorporated in the cell is recovered and then measured using ascintillator, and the incorporation of the ³H thymidine into the cell isused as an indicator to assay the immunosuppressive activity.

Examples of such an antibody include an antibody produced from thehybridoma HD4 (accession number: FERM BP-7771), an antibody producedfrom the hybridoma HD8 (accession number: FERM BP-7773), and an antibodyproduced from the hybridoma HD10 (accession number: FERM BP-7774).

The aforementioned property a) indicates that the antibody has potentanti-tumor activity.

The antibody according to the present invention has a functionalproperty, that is, activity of suppressing immune responses byallogeneic mixed lymphocyte reaction is equivalent to or higher thanthat of L243. More specifically, the immunosuppressive activity isequivalent to or higher than that achieved when using the mouseanti-HLA-DR monoclonal antibody L243 (ATCC HB-55) at the sameconcentration, wherein the immunosuppressive activity is assayed asfollows. First, 50 μl of antibody adjusted at 8–200 μg/mL, preferably 8μg/mL using 10% FCS-containing RPMI 1640 medium is mixed with 50 μL ofmature dendritic cell suspension derived from a first human donoradjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium inwells of a 96-well plate. The mixture is then allowed to stand at 4° C.for 30 minutes, and the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor.The resultant is cultured at 37° C. in the presence of 5% CO₂ for 5days, ³H thymidine is added thereto at 1.0 μCi/well, the resultant isfurther cultured at 37° C. in the presence of 5% CO₂ for 16 to 20 hours,the ³H thymidine incorporated in the cell is recovered and then measuredusing a scintillator, and incorporation of the ³H thymidine into thecell is used as an indicator to assay the immunosuppressive activity. Anexample of such an antibody is one produced from the hybridoma HD6(accession number: FERM BP-7772).

The antibody having the aforementioned activity according to the presentinvention is useful as an ingredient for a preventive or therapeuticagent for malignant tumors or an ingredient for an immunosuppressiveagent.

Even more surprisingly, the antibody according to the present inventionsignificantly suppress a lowering in the survival ratio of tumor-bearingmouse models caused by the tumor cell growth at a low dose of 0.1 μg permouse (5 μg per kg of body weight), and exhibits life extending-effectsin mouse models. If the survival ratio of the mice to which the antibodyof the present invention has been administered is significantly enhancedcompared with that of the control mice when the human anti-human serumalbumin (HSA) antibody is administered as a control simultaneously withthe antibody according to the present invention, the antibody of thepresent invention can be determined to exhibit the lifeextending-effects. For example, the antibody according to the presentinvention and the anti-HSA antibody are administered to 5 each oftumor-bearing mouse models to which lymphoma cells have beentransplanted. If at least one mouse to which the antibody of the presentinvention has been administered is alive when all of the mice in thegroup of mice to which the anti-HSA antibody has been administered havedied, it can be said that the life extending-effects can be exhibited intumor-bearing mouse models.

The immunosuppressive effects can be evaluated based on activity ofsuppressing the immune response by allogeneic mixed lymphocyte reaction(MLR) as described above, and MLR can be carried out in a conventionalmanner.

Also, the epitope of the HLA-DR recognized by the antibody of thepresent invention can be identified by a conventional method. Forexample, regarding 199 amino acids in the extracellular region of theHLA-DR β chain (DRB1*15011) (199 amino acids from amino acids 29 to 227in the amino acid sequence as shown in SEQ ID NO: 147, and thenucleotide sequence encoding the amino acid sequence as shown in SEQ IDNO: 147 is shown in SEQ ID NO: 146), peptides are prepared by shiftingan amino acid in the 13-mer peptides (for example, peptides having aminoacid sequences as shown in SEQ ID NOs: 52 to 145), and the reactivity isinspected. In such a case, the antibody according to the presentinvention maximally binds to the peptide having the amino acid sequenceas shown in SEQ ID NO: 82, or potently binds to at least one of thethree peptides as shown in SEQ ID NOs: 82, 83, and 84. The term“potently binds” refers to the binding which exhibits fluorescenceintensity at least 10 times as great as that of the background in themethod as described in Example 17 (2). The antibody of the presentinvention has reactivity with peptides 61 to 71 of the HLA-DR β chain.Further, HLA-DR is known to have approximately 350 types ofpolymorphisms (see the IMGT/HLA database of EMBL-EBI, etc.). Theantibody according to the present invention is capable of recognizingsubstantially almost all of these polymorphisms. Whether or not this istrue can be determined by preparing a group of peptides includingsubstantially almost all of the approximately 350 types of polymorphismsand assaying the reactivity with substantially almost all of thesepeptides. For example, if an antibody reacts with 12 or more types ofpeptides among 16 types of peptides as shown in SEQ ID NOs: 24 to 39,this antibody is capable of recognizing substantially almost all of thepolymorphisms of the HLA-DR. The antibody according to the presentinvention significantly binds to all the peptides as shown in SEQ IDNOs: 24 to 39 and all the peptides as shown in SEQ ID NOs: 40 to 43. Theterm “significantly binds” refers to the binding which exhibitsfluorescence intensity at least 10% as great as the background in themethod as described in Example 17 (3).

2. Pharmaceutical Composition

A preparation that comprises a purified preparation of the humananti-HLA-DR antibody of the present invention is also within the scopeof the present invention. Such a preparation preferably comprises aphysiologically acceptable diluent or carrier in addition to theantibody, and it may be a mixture of the aforementioned antibody withanother antibody or another agent such as an antibiotic. Examples of asuitable carrier include, but are not limited to, physiological saline,phosphate-buffered saline, phosphate-buffered saline glucose solution,and a buffered saline solution. Alternatively, the antibody may belyophilized and used by being recomposed with the addition of the abovebuffered aqueous solution, when it is needed. The preventive ortherapeutic agent can be administered in various dosage forms, andexamples of dosage forms include oral administration in the form of, forexample, a tablet, capsule, granule, powder, or syrup and parenteraladministration in the form of, for example, an injection, drop, orsuppository.

The dose may vary depending on symptom, age, body weight, etc. In thecase of oral administration, the dose is generally about 0.01 mg to1,000 mg per day per adult, and this amount of preparation can beadministered in one dose or several separate doses. In the case ofparenteral administration, about 0.01 mg to 1,000 mg can be administeredper dose through hypodermic injection, intramascular injection, orintravenous injection.

The antibody or a pharmaceutical composition according to the presentinvention can be applied to the treatment or prevention of variousdiseases or symptoms that could be caused by an HLA-DR-expressing cell.Examples of such diseases or symptoms include various malignant tumors,and examples of the application thereof are use as an immunosuppressiveagent at the time of organ transplantation (a preventive or therapeuticagent for immunological rejection at the time of pancreatic islet orkidney transplantation, or GVHD), a therapeutic agent for autoimmunediseases (for example, rheumatism, arteriosclerosis, multiple sclerosis,systemic erythematodes, idiopathic thrombocythemia, or Crohn's disease),and a therapeutic agent for allergic diseases such as asthma.

For example, the antibody according to the present invention and apharmaceutical composition comprising this antibody having the followingfunctional properties a) and b) can be used for preventing or treatingvarious malignant tumors:

a) HLA-DR-expressing human cancer cells are transplanted inimmunodeficient mice such as SCID mice, the survival ratio of mice wheninoculated with the antibody of the present invention is inspected, andas a result, the number of days for which the mice survive is prolonged;and

b) activity of suppressing immune responses by allogeneic mixedlymphocyte reaction is lower than that of L243.

The antibody according to the present invention and a pharmaceuticalcomposition comprising this antibody having activity of suppressingimmune responses by allogeneic mixed lymphocyte reaction equivalent toor higher than that of L243 can be used for preventing or treatingrheumatism or graft-versus-host disease (GvHD).

The present invention includes a process for preventing or treating theaforementioned diseases using the antibody or pharmaceutical compositionaccording to the present invention. The present invention also includesthe use of the antibody according to the present invention in theproduction of a preventive or therapeutic agent for the aforementioneddiseases.

Examples of tumors that can be prevented or treated are leukemia(including chronic lymphatic leukemia and acute lymphatic leukemia),lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-celllymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma,diffuse lymphoma, and follicular lymphoma), myeloma (including multiplemyeloma), breast cancer, colon cancer, kidney cancer, gastric cancer,ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer,esophageal cancer, liver cancer, head and neck squamous cancer, skincancer, urinary tract cancer, prostate cancer, choriocarcinoma,pharyngeal cancer, laryngeal cancer, pleural tumor, arrhenoblastoma,endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi'ssarcoma, angioma, cavernous angioma, hemangioblastoma, retinoblastoma,spongiocytoma, neurofibroma, oligodendroglioma, medulloblastoma,neuroblastoma, neuroglioma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor. When theantibody of the present invention is applied, the kind of a tumor is notlimited to one, and combinations of several kinds of tumors may beinvolved.

3. Preparation Example

The molecule according to the present invention is used as an ampule foran aseptic solution or suspension dissolved in water or anotherpharmaceutically acceptable solution. Also, the ampule is filled with anaseptic powder preparation (preferably lyophilized molecules of thepresent invention), and it may be diluted with a pharmaceuticallyacceptable solution at the time of use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the immunosuppressive activity of thepurified human anti-HLA-DR monoclonal antibody on MLR.

FIG. 2 is a diagram showing the life-extending effects of the purifiedhuman anti-HLA-DR monoclonal antibody on the tumor-bearing mouse model.FIG. 2A shows a case where the dose is 1 μg/head, and FIG. 2B shows theeffects attained when the dose is 0.1 μg/head.

FIG. 3 is a diagram showing the antibody-dependent cellular cytotoxicity(ADCC) and the complement-dependent cytotoxicity (CDC) of the purifiedhuman anti-HLA-DR monoclonal antibody. FIG. 3A shows the ADCC activitiesof HD8G1Ser and HD8G1, FIG. 3B shows the CDC activities of HD8G2CHO,HD8G1, HD8G1Ser, and HD8G4, FIG. 3C shows the ADCC activities of HD8G1,HD8G2, HD8G2Ser, HD8G4, HD4G1, HD4G2Ser, and HD4G4, and FIG. 3D showsthe CDC activities of HD8G1, HD8G2, HD8G2Ser, HD8G4, HD4G1, HD4G2Ser,and HD4G4.

FIG. 4 is a diagram showing the life-extending effects of the antibodiesaccording to the present invention, HD8G1Ser, HD8G2Ser, and HD8G1, onthe tumor-bearing mouse model. FIG. 4A shows the results attained whenthe dose is 0.1 μg per mouse, and FIG. 4B shows the results attainedwhen the dose is 1.0 μg per mouse.

FIG. 5 is a diagram showing the analysis of the purified humananti-HLA-DR monoclonal antibody epitope using the synthetic peptide inthe HLA-DR β chain sequence. FIGS. 5A, 5B, and 5C show analyses on HD4,HD6, and HD8, respectively.

FIG. 6 is a diagram showing the analysis of the purified humananti-HLA-DR monoclonal antibody epitope using the synthetic peptide inthe polymorphic sequence in the HLA-DR β chain epitope sequence.

FREE TEXT OF SEQUENCE LISTING

-   SEQ ID Nos: 1 to 15: description of artificial sequence: primer-   SEQ ID Nos: 24 to 145: description of artificial sequence: peptide

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail withreference to the following examples, although the technical scope of thepresent invention is not limited to the embodiments described in theseexamples.

EXAMPLE 1 Preparation of Antigen

In order to obtain a cell having excessively expressed human HLA-DR onits cell membrane, plasmid vectors for the expression of full-lengthamino acids in the human HLA-DR α chain and β chain were prepared. DNAencoding the HLA-DR α chain and β chain was prepared by PCR.

a) Preparation of Expression Vectors for Full-length Human HLA-DR αChain and β Chain

In template PCR, plasmid vectors pEF-neo-HLA-DRα and pEF-neo-HLA-DRβholding cDNA encoding the human HLA-DR α chain and β chain were used asthe templates. pEF-neo-HLA-DRα and pEF-neo-HLA-DRβ were prepared in themanner as described below. DNA of the full-length human HLA-DR α chainand that of the HLA-DR β chain were modified by the polymerase chainreaction (PCR) for adding the EcoRI sequence at the 5′-terminus and theNotI sequence and a stop codon at the 3′-terminus. PCR was carried outfor 30 PCR cycles of 94° C. for 15 seconds, 55° C. for 30 seconds, and72° C. for 60 seconds using, as a template, the cDNA derived from humanperipheral blood mononuclear cells, as primers, synthesizing5′-CCGGAATTCCCACCATGGCCATAAGTGGAGTCCCTGTG-3′ (SEQ ID NO: 1) with5′-AAAGCGGCCGCTCATFACAGAGGCCCCCTGCGTTCTGC-3′ (SEQ ID NO: 2) for theHLA-DRα and synthesizing 5′-CCGGAATTCCTGGTCCTGTCCTGTTCTCCAGCA-3′ (SEQ IDNO: 3) with 5′-AAAGCGGCCGCTCATCAGCTCAGGAATCCTGTTGGCTG-3′ (SEQ ID NO: 4)for the HLA-DRβ, and the LA-Taq DNA polymerase (Gibco BRL). Thesynthesized sequence was isolated as the EcoRI-NotI fragment and linkedto the pEF-neo vector cleaved with the same enzyme (a vector comprisingthe neomycin-resistant gene incorporated in modified pEF-BOS (seeMizushima S. & Nagata S., Nucleic Acids Res (1990), 18, 5332)). Theresulting plasmids were designated as pEF-neo-HLA-DRα andpEF-neo-HLA-DRβ. 765 bp cDNA was encoded in the HLA-DRα incorporated inpEF-neo-HLA-DRα, and 801 bp cDNA was encoded in the HLA-DRβ incorporatedin pEF-neo-HLA-DRβ. In all of PCRs in the following examples, thereaction temperature was regulated using the Gene Amp PCR System 9700(Perkin Elmer Japan).

c) Preparation of a Human HLA-DR-expressing Cell

pEF-neo-HLA-DRα and pEF-neo-HLA-DRβ prepared in b) were introduced inthe L929 cell (American Type Culture Collection (ATCC) No. CCL-1) usingLipofectAMINE Plus (Gibco BRL). Gene introduction was carried out inaccordance with the method described in the instructions therefor. Thegene-introduced cells were cultured in a cell culture flask (culturearea: 75 cm²) at 37° C. in the presence of 5.0% CO₂ for 24 hours, andG418 (Gibco BRL) was added thereto at 1 mg/mL, followed by culture for aweek. Subsequently, flow cytometry (FCM, Becton Dickinson) was carriedout using the R-phycoerythrin-labeled mouse anti-human-HLA-DR antibody(BD Pharmingen), and cells having HLA-DR expressed on the surfaces oftheir cell membranes were selectively sorted from those that hadattained G418-resistance among the gene-introduced cells.

All oligonucleotides such as PCR primers were synthesized using a DNAautomatic synthesizer (Model: 3948, Perkin-Elmer's Applied BiosystemsDivision) in accordance with the attached instructions (see Matteucci,M. D. and Caruthers, M. H., (1981), J. Am. Chem. Soc. 103, 3185–3191).After the completion of the synthesis, each of the oligonucleotides wascleaved from the support and then deprotected. The resulting solutionwas exsiccated, dissolved in distilled water, and then cryopreserved at−20° C. before use.

EXAMPLE 2 Preparation of Human Antibody-producing Mouse

The mouse used for immunization is genetically homozygous for bothendogenous Ig heavy chain breakdown and κ light chain breakdown, andsimultaneously retains a chromosome 14 fragment (SC 20) containing ahuman Ig heavy chain locus and a human Igκ chain transgene (KCo5). Thismouse was prepared by mating a mouse having a human Ig heavy chain locus(lineage A) with a mouse having a human Igκ chain transgene (lineage B).Lineage A is homozygous for both endogenous Ig heavy chain breakdown andκ light chain breakdown, and retains a chromosome 14 fragment (SC20)that is transmittable. For example, it is described in the report byTomizuka et al. (Tomizuka et al., Proc. Natl. Acad. Sci. USA, 2000, Vol.97: 722). Lineage B is homozygous for both endogenous Ig heavy chainbreakdown and κ light chain breakdown, and retains a human Igκ chaintransgene (KCo5, a transgenic mouse). It is described for example, inthe report by Fishwild et al. (Nat. Biotechnol., 1996, Vol. 114: 845).

An individual obtained by mating a male mouse of lineage A with a femalemouse of lineage B or a female mouse of lineage A with a male mouse oflineage B in which the human Ig heavy chain and the κ light chain can besimultaneously detected in the serum (Ishida & Lonberg, IBC's 11thAntibody Engineering, Abstract 2000) was used in the following immunityexperiment. The aforementioned human antibody-producing mouse (referredto as a “KM mouse”) can be obtained from Kirin Brewery Co., Ltd. bymaking a contract.

EXAMPLE 3 Preparation of Human Monoclonal Antibody Against Human HLA-DR

In this example, a monoclonal antibody was prepared in accordance with acommon technique as described in, for example, “Tan-kuron koutai jikkensousa nyuumon (A guide to monoclonal antibody experiments)” (Tamie ANDOet al., Kodansha Ltd. Publishers, 1991). The HLA-DR-expressing L929 cellprepared in Example 1 was used as the immunogen human HLA-DR. A humanimmunoglobulin-producing human antibody-producing mouse prepared inExample 2 was used as an animal to be immunized.

In order to prepare a human monoclonal antibody against human HLA-DR,human antibody-producing mice were subjected to initial immunizationintraperitoneally with the HLA-DR-expressing L929 cells (5×10⁶ cells permouse) prepared in Example 1. After the initial immunization, the samecells were administered for immunization 2, 4, and 8 weeks later. Threedays before obtaining the spleen and the lymph node as described below,cells (1×10⁶ cells per mouse) were administered to the tail veins, andrecombinant human IL-6 (hereafter referred to as “IL-6,” 5 ng per mouse,prepared at the Pharmaceutical Research Laboratory, Kirin Brewery Co.,Ltd.) was administered subcutaneously.

The spleen and/or lymph node were surgically obtained from the immunizedmouse, the recovered organs were placed in 10 mL of serum-free DMEMmedium containing 350 mg/mL sodium bicarbonate, 50 units/mL ofpenicillin, and 50 μg/mL of streptomycin (Gibco BRL, hereafter referredto as “serum-free DMEM medium”), and mashed on a mesh (cell strainer,Falcon) using a spatula. The cell suspension that had passed through themesh was centrifuged to precipitate the cells. Thereafter, these cellswere washed twice in serum-free DMEM medium and then suspended inserum-free DMEM medium to count the number of cells. In contrast,myeloma cells SP2/0 (ATCC No. CRL-1581), which were cultured in DMEMmedium (Gibco BRL) containing 10% FCS (SIGMA) (this medium is hereafterreferred to as “serum-containing DMEM medium”) at 37° C. in the presenceof 5% CO₂ in such a manner that the cell concentration did not exceed1×10⁶ cells/mL, were similarly washed in serum-free DMEM medium and thensuspended in serum-free DMEM medium to count the number of cells. Therecovered cell suspension and the mouse myeloma cell suspension weremixed with each other at a ratio of 5:1, the mixture was centrifuged,and the supernatant was completely removed. 1 mL of 50% (w/v)polyethylene glycol 1500 (Boehringer Mannheim) was slowly added as afusing agent to the pellet while stirring the pellet with the tip of apipette, 1 mL of serum-free DMEM medium previously heated at 37° C. wasslowly added two separate times, and 7 mL of serum-free DMEM medium wasfurther added. After the centrifugation, the supernatant was removed,and the resulting fusion cell was subjected to screening by limitingdilution as described below. A hybridoma was selected by culturing it inDMEM medium containing 10% FCS, IL-6 (10 ng/mL), and hypoxanthine (H),aminopterin (A), and thymidine (I) (hereafter referred to as “HAT,”SIGMA). Further, a single clone was obtained by limiting dilution usingHT (SIGMA), 10% FCS, and IL-6-containing DMEM medium. Culture wasconducted in a 96-well microtiter plate (Becton Dickinson). Theselection (screening) of a hybridoma clone that produces the anti-humanHLA-DR human monoclonal antibody and the characterization of the humanmonoclonal antibody that is produced by each hybridoma were carried outwith enzyme-linked immunosorbent assay (ELISA) and FCM as describedlater.

In the screening of human monoclonal antibody-producing hybridomas, manyhuman monoclonal antibody-producing hybridomas were obtained, which havehuman immunoglobulin γ chain (hIgγ) and human immunoglobulin light chainκ, and which have specific reactivity with human HLA-DR through CellELISA as described in Examples 4 and 5.

EXAMPLE 4 Selection of Human Anti-HLA-DR Monoclonal Antibody-producingClones Having Human Immunoglobulin Light Chain κ (Igκ)

Burkitt's lymphoma cells, Daudi (ATCC No. CCL-213), were added to eachwell in quantities of 1×10⁵ cells, the hybridoma supernatant was addedthereto, and the mixture was incubated at 4° C. for 20 minutes.Subsequently, the incubation product was washed twice with 1%FCS-containing PBS, the horseradish peroxidase-labeled goat anti-humanimmunoglobulin light chain κ (Igκ) antibody (50 μg/well, DAKO) was addedthereto, and the resultant was incubated at 4° C. for 20 minutes. Theincubation product was washed twice with 1% FCS-containing PBS, and 100μL each of a TMB chromogenic substrate solution (DAKO) was added to eachwell, followed by incubation at room temperature for 20 minutes. 0.5Msulfuric acid (100 μL/well) was added to each well to terminate thereaction. Absorbance at a 450 nm wavelength (reference wavelength: 570nm) was assayed using a microplate reader (1420 ARVO multilabel counter,Wallac) to select positive antibody-producing clones.

EXAMPLE 5 Identification of Subclass of Each Monoclonal Antibody

After the addition of 1×10⁵ Daudi cells to each well, the hybridomasupernatant was added thereto, and the resultant was incubated at 4° C.for 20 minutes. Subsequently, the incubation product was washed twicewith 1% FCS-containing PBS, and horseradish peroxidase-labeled sheepanti-human IgG1 antibody, sheep anti-human IgG2 antibody, sheepanti-human IgG3 antibody, or sheep anti-human IgG4 antibody (2000-folddiluted, 50 μL/well, The Binding Site) was added to each well, followedby incubation at room temperature for 1 hour. After being washed threetimes with 1% FCS-containing PBS, a substrate buffer (TMB, 100 μL/well,DAKO) was added to each well, and incubation was carried out at roomtemperature for 20 minutes. Subsequently, 0.5M sulfuric acid (100μL/well) was added to terminate the reaction. Absorbance at a 450 nmwavelength (reference wavelength: 570 nm) was assayed using a microplatereader (1420 ARVO multilabel counter, Wallac) to identify the subclassfor each clone. Clones which were not positive for any subclass wereexcluded from the selection since they were not IgG The results only onthe finally selected clones are shown in Table 1.

TABLE 1 Properties of selected anti-HLA-DR monoclonal antibodiesActivity of suppressing immune responses by Reactivity allogeneic mixedReactivity with L929/ Subclass lymphocyte reaction L929 HLA-DR Human IgGPoly − − − HD3 IgG1 ++ − + HD4 IgG1 ++ − + HD6 IgG1 +++ − + HD7 IgG3 +− + HD8 IgG2 + − + HD10 IgG2 + − +

EXAMPLE 6 Process for Obtaining Normal Human Mononuclear Cell and NormalHuman Dendritic Cell

At the outset, a normal human peripheral blood-derived mononuclear cellwas prepared in accordance with a conventional method using Ficoll(Ficoll-PaquePLUS, Amersham Pharmacia Biotech). The normal human bloodcontained in a blood-sampling bag (Terumo) containing a sodium citratesolution as an anticoagulant was centrifuged (600 G, room temperature, 5minutes) to separate a cell fraction from blood plasma. The cellfraction was diluted with PBS, superposed on Ficoll, and mononuclearcells were separated by specific gravity-based centrifugation (400 G,room temperature, 30 minutes). The intermediate layer was extracted as amononuclear cell and washed twice with PBS. The resultant was furtherdiluted with PBS, and centrifuged at 100 G for 10 minutes to removeblood platelets remaining in the supernatant. Thus, normal humanperipheral blood-derived mononuclear cells (PBMC) were obtained.

Subsequently, the obtained PBMC was allowed to react with CD14 antibodyattached to magnetic beads (Miltenyi Biotec (MB)) at 4° C. for 30minutes, and positive selection for CD14-positive cells was carried outthrough a MACS separating column (MB). The MACS separating column wasused in accordance with the attached instruction. The CD14-positivecells were cultured in 10% FCS-containing RPMI medium comprising GM-CSF(final concentration: 50 ng/mL, prepared at the Pharmaceutical ResearchLaboratory, Kirin Brewery Co., Ltd.) and interleukin 4 (finalconcentration: 200 ng/mL, Genzyme Corporation) for 5 to 8 days.Thereafter, lipopolysaccharide (LPS, final concentration: 40 ng/mL,Difco) was added thereto, and the resultant was cultured overnight. Theculture product was then used as a mature dendritic cell (mature DC).

EXAMPLE 7 Activity of Suppressing Immune Responses by Allogeneic MixedLymphocyte Reaction

In an allogeneic transplantation having different majorhistocompatibility antigens (MHC), the T-cell is activated byrecognizing the nonself (histoincompatible) MHC molecular complex(alloantigen), thereby generating immunological rejection. The human MHCis referred to as human leukocyte antigen (HLA), and there are class Iantigens to which HLA-A, B, and C belong, and class II antigens to whichHLA-DP, DQ, and DR belong. Further, since each molecule has apolymorphic property, several thousand combinations of human HLA arepossible. Thus, histoincompatibility is very highly likely to occur withanother person. The allogeneic mixed lymphocyte culture is a test toinspect in vitro the growth of T-cells which react with alloantigens bysubjecting lymphocytes having different histocompatible antigens(hereafter referred to as donor A and donor B for convenience) to mixedculture.

Activity of suppressing the immune response by allogeneic mixedlymphocytes was assayed using the culture supernatant of hybridomasselected in Examples 4 and 5. A normal human peripheral bloodmononuclear cell was obtained in the manner as described in Example 6.The mononuclear cells of donor A were allowed to react in RPMI 1640medium containing mitomycin C (25 μg/mL, 37° C., 30 minutes) to suppressthe multiplication of cells. After the reaction, they were washed atleast three times in RPMI 1640 medium and suspended in RPMI 1640 mediumat 1×10⁶/mL. The mononuclear cells of donor B were fractionated in a96-well plate at 1×10⁵ cell/well, the medium was removed bycentrifugation, the culture supernatant was added at 100 μL/well, andthe resultant was allowed to stand at 4° C. for 30 minutes.

Subsequently, the mononuclear cells of donor A were fractionated at 100μL/well in a 96-well plate containing the mononuclear cells of donor Band the culture supernatant, followed by culturing at 37° C. for 4 daysin the presence of 5% CO₂. Thereafter, ³H thymidine (Amersham PharmaciaBiotech) was added thereto at 1.0 μCi/well, and culture was furtherconducted at 37° C. for 16 to 20 hours in the presence of 5% CO₂. The ³Hthymidine incorporated in cells were collected on a glass filter mat(Printed Filtermat, Wallac) using the Micro96 Harvester (SKATRON),dehydrated, well immersed in scintillator (Betap, Scint, Wallac), andpackaged. Thereafter, activity of the β dose was assayed using a liquidscintillation counter (1205 BETAPLATE, Wallac).

Properties of the anti-human HLA-DR antibodies selected as a result ofthe assay are shown in Table 1. Among those, HD8, HD10, HD4, and HD6 aredeposited internationally at the International Patent OrganismDepositary of the National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)as of Oct. 11, 2001. The accession number of the hybridoma HD8 is FERMBP-7773, that of the hybridoma HD10 is FERM BP-7774, that of thehybridoma HD4 is FERM BP-7771, and that of the hybridoma HD6 is FERMBP-7772.

EXAMPLE 8 Preparation of Each Antibody

Thu human anti-HLA-DR monoclonal antibody was purified from the culturesupernatant of hybridomas obtained in Examples 4 and 5 in the followingmanner. The culture supernatant containing the human anti-HLA-DRmonoclonal antibody was subjected to affinity purification using rmpProtein A (Amersham Pharmacia Biotech) and a 0.8×4.0 cm column (Biorad),PBS as an adsorption buffer, and a 0.02M glycin buffer (pH 3) as anelution buffer. The elution fraction was adjusted at around pH 7.2 withthe addition of 1M Tris (pH 9.0). The prepared antibody solution wasconverted into PBS using a dialysis membrane (molecular weight cutoff:10,000, Spectrum Laboratories), filter-sterilized with a membrane filterMILLEX-GV (pore diameter: 0.22 μm, MILLIPORE), and the purified humananti-HLA-DR monoclonal antibody was obtained. The concentration of thepurified antibody was calculated by assaying the absorbance at 280 nmand on the basis of 1 mg/mL=1.4 OD.

The culture supernatant containing the human anti-HLA-DR monoclonalantibody was prepared in the following manner. At the outset, the humananti-HLA-DR monoclonal antibody-producing hybridoma was adapted to eRDFmedium (Kyokuto Seiyaku) containing 10 ng/ml of IL-6 and 10% fetal calfserum (FCS, SIGMA). Subsequently, a part thereof was adapted to eRDFmedium (Kyokuto Seiyaku) containing bovine insulin (5 μg/ml, Gibco BRL),human transferin (5 μg/ml, Gibco BRL), ethanolamine (0.01 mM, SIGMA),sodium selenite (2.5×10⁻⁵ mM, SIGMA), and 1% low IgG FCS (HyClone) topurify the antibody. These adapted hybridomas were cryopreserved.Culture was conducted in a flask, and the culture supernatant wascollected when the rate of surviving hybridomas reached 90%. Thecollected supernatant was applied to 10 μm- and 0.2 μm-filters (GelmanScience) to eliminate waste such as hybridomas.

EXAMPLE 9 Activity of Suppressing Immune Responses by Allogeneic MixedLymphocytes by Purified Human Anti-HLA-DR Monoclonal Antibody

The allogeneic mixed lymphocyte culture is a test to inspect in vitrothe growth of T-cells which react with alloantigens by subjectinglymphocytes having different histocompatible antigens (hereafterreferred to as donor A and donor B for convenience) to mixed culture.The mature dendritic cell (DC) derived from a monocyte in vitro inducedfrom the peripheral blood of donor A and the T-cells separated from theperipheral blood of donor B are exclusively subjected to mixed culture.This enables the inspection of similar reactivity of T-cells with analloantigen when the antibody-dependent cellular cytotoxicity (ADCC) ofthe macrophage, etc. has been removed. Mixed culture using allogeneic DCand T-cells was carried out in the presence of the anti-HLA-DRmonoclonal antibody to inspect the function of the anti-HLA-DRmonoclonal antibody on the T-cell alloantigen reactivity. Specifically,the mature DC of donor A was suspended in 10% FCS-containing RPMI 1640medium (RPMI-10% FCS) at 2×10 cells/mL on a round-bottom, 96-well plate(Falcon). The anti-HLA-DR monoclonal antibody, the human polyclonal IgG(hIgG) as a negative control, and the mouse anti-HLA-DR monoclonalantibody L243 (ATCC HB-55) as a positive control were diluted withRPMI-10% FCS to 200, 40, and 8 μg/mL, respectively. The T-cellsseparated from the peripheral blood of donor B (purity: 99% or higher)were suspended in RPMI-10% FCS at 1×10⁶ cells/mL. At the outset, 50 μLof the mature DC derived from donor A was mixed with the same amount ofthe anti-HLA-DR monoclonal antibody on a 96-well plate, and theresultant was allowed to stand at 4° C. for 30 minutes. Subsequently,100 μL of T-cells derived from donor B was mixed therewith, cultured at37° C. for 5 days in the presence of 5% CO₂, ³H thymidine (AmershamPharmacia Biotech) was added at 1.0 μCi/well, and culture was furtherconducted at 37° C. for 16 to 20 hours in the presence of 5% CO₂. The ³Hthymidine incorporated in the cells was collected on a glass filter mat(Printed Filtermat, Wallac) using the Micro96 Harvester (SKATRON),dehydrated, well immersed in a scintillator (Betap, Scint, Wallac), andpackaged. Thereafter, activity of the jβ-ray dose was assayed using aliquid scintillation counter (1205 BETAPLATE, Wallac).

The results are shown in FIG. 1. The immunosuppressive activities ofHD4, HD6, HD8, and HD10 were more dose-dependent compared with those ofthe hIgG group, those of HD8, HD4, and HD10 were lower than those ofL243, and those of HD6 were equivalent to or higher than those of thepositive control antibody L243.

This suggests that HD8, HD4, and HD10 can be used as antibodies havinglow immunosuppressive activities, and HD6 can be used as animmunosuppressive agent.

EXAMPLE 10 Examination of Reactivity of Each Monoclonal Antibody withHLA-DR-expressing Cells

Reactivity of each purified monoclonal antibody obtained in Example 3with the HLA-DR-expressing L929 cells prepared in Example 1 was analyzedby FCM. The L929 cells and the HLA-DR-expressing L929 cells weresuspended in PBS containing 0.1% sodium azide and 1% fetal calf serum(Staining Medium, hereinafter abbreviated to “SM”) at 2×10⁷/mL, and thesuspension was fractionated in a 96-well, round-bottom plate at 100μl/well. After the centrifugation (600 G, 4° C., 2 minutes), thesupernatant was removed, the culture supernatant (50 μl) of hybridomascultured in Example 3 was added, and the mixture was stirred and allowedto stand under ice cooling for 30 minutes. Centrifugation (600 G, 4° C.,2 minutes) was then carried out to remove the supernatant. The pelletwas washed twice with 100 μl/well SM, 30 μL of 0.0125 mg/mL RPEfluorescence-labeled rabbit anti-human Igκ F(ab′)₂ antibody (DAKO) wasadded thereto, and the resultant was incubated under ice cooling for 30minutes. After being washed twice with SM, the incubation product wassuspended in SM, and the fluorescence intensity of each cell was assayedby FCM.

The results are shown in Table 1 above. All the antibodies exhibitedpotent binding activities to HLA-DR-expressing L929 cells alone, butnone exhibited the binding activity to the L929 cell. This indicatesthat these antibodies specifically bind to HLA-DR.

EXAMPLE 11 Effect of Purified Human Anti-HLA-DR Monoclonal Antibody onTumor-bearing Mouse Models

The effects of the purified human anti-HLA-DR monoclonal antibodyobtained in Example 8 were examined using tumor-bearing mouse models inaccordance with a method described below.

First, 5-week-old C.B-17/ICR SCID mice (CLEA Japan, Inc.) werepurchased, anti-asialo GM1 antiserum (Wako Chemicals) was diluted, and10 μL each thereof was intravenously administered to each of the micewhen they were 6 weeks old. On the next day, Burkitt's lymphoma cellsRaji (ATCC CCL-86) were intravenously administered in amounts of 5×10⁶cells per mouse. Five days after the Raji transplantation, the purifiedhuman anti-HLA-DR monoclonal antibody was administered once in tailveins of mice in amounts of 0.1 μg or 1 μg per mouse. As an antibodynegative control, the same amount of human anti-HSA antibody was used.The survival ratio after the transplantation was observed for about 3months or longer.

The results of the experiment are shown in FIG. 2. In the group of miceto which the purified human anti-HLA-DR monoclonal antibody wasadministered in amounts of 0.1 μg per mouse, the number of survivingmice 90 days later was 3 for HD8, and 1 for HD10 and HD4 out of a groupof 5 mice (FIG. 2B). In contrast, in the group of mice to which theantibody was administered in amounts of 1.0 μg per mouse, the number ofsurviving mice was 3 for HD8, 5 for HD10, and 2 for HD 4 out of a groupof 5 mice (FIG. 2A). In the negative control group to which the anti-HSAantibody was administered, all mice died within 60 days after the Rajitransplantation.

At the time of the antibody administration, the body weight of eachmouse was about 20 g. Thus, 0.1 μg and 1 μg per mouse means 5 μg/kg and50 μg/kg based on body weight, respectively. This revealed that HD8exhibits an anti-tumor effect at very low dosages. Based on this and theresults attained in Example 9 together, HD8 can be said to be anantibody having low immunosuppressive activity and potent anti-tumoractivity. This suggests that HD8 can be used as an anti-cancer agentwith few side effects.

EXAMPLE 12 Preparation of a Gene Encoding a Monoclonal Antibody andConstruction of a Recombinant Antibody-expressing Vector

(1) cDNA Cloning of HD4 and HD8 Antibody Genes and Preparation ofExpression Vector

Hybridomas HD4 and HD8 were cultured in eRDF medium (Kyokuto Seiyaku)containing 10 ng/ml of IL-6 (R & D Systems) and 10% fetal bovine serum(SIGMA) and centrifuged to collect cells. Thereafter, TRIZOL (Gibco BRL)was added thereto, and total RNA was extracted in accordance with theinstructions therefor. Cloning of the variable region in the antibodycDNA was carried out using the SMART RACE cDNA amplification Kit(Clontech) in accordance with the attached instruction.

First-strand cDNA was prepared using 5 μg of the total RNA as atemplate.

1) Synthesis of First-strand cDNA

-   -   Total RNA 5 μg/3 μL    -   5′CDS 1 μL    -   SMART oligo 1 μL

The reaction solution having the above composition was incubated at 70°C. for 2 minutes,

-   -   5× Buffer 2 μL    -   DTT 1 μL    -   DNTP mix 1 μL    -   Superscript II 1 μL        were then added, and the mixture was incubated at 42° C. for 1.5        hours.

Further, 100 μL of Tricine Buffer was added, followed by incubation at72° C. for 7 minutes. Thus, fist-strand cDNA was obtained.

2) Amplification of a Heavy Chain Gene and a Light Chain Gene by PCR andConstruction of a Recombinant Antibody-expressing Vector

cDNA was amplified using Z-Taq (Takara).

-   -   cDNA 2 μL    -   10×Z-Taq Buffer 5 μL    -   dNTP mix 4 μL    -   Z-Taq 1 μL    -   Primer 1    -   Primer 2

The final volume of the reaction solution having the above compositionwas brought to 50 μL with the aid of double distilled water and thensubjected to PCR.

The heavy chain was amplified by 30 PCR cycles of 98° C. for 1 secondand 68° C. for 30 seconds using UMP (SMART RACE cDNA amplification Kit,Clontech) and the hh-6 primer (5′-GGT CCG GGA GAT CAT GAG GGT GTCCTT-3′) (SEQ ID NO: 5). Further, 1 μL of this reaction solution was usedas a template, and 20 PCR cycles of 98° C. for 1 second and 68° C. for30 seconds were repeated using NUMP (SMART RACE cDNA amplification Kit,Clontech) and the hh-3 primer (5′-GTG CAC GCC GCT GGT CAG GGC GCC TG-3′)(SEQ ID NO: 6). Thereafter, the amplified PCR product was purified usinga PCR purification kit (QIAGEN), and nucleotide sequencing was carriedout using hh-4 (5′-GGT GCC AGG GGG AAG ACC GAT GG-3′) (SEQ ID NO: 7) asa primer. Based on the sequence information, the HD4 heavy chainspecific primer tnHD4Sal (5′-ata tgt cga cCC AGC CCT GGG ATT TTC AGG TGTTTT C-3′) (SEQ ID NO: 8) and the HD8 heavy chain specific primertnHD8Sal (5′-ata tgt cga cTGG CTG ACC AGG GCA GTC ACC AGA G-3′) (SEQ IDNO: 9) were synthesized, and the resultant primer was used to determinethe sequence also from the opposite direction. PCR was carried out usinga specific primer and tnCHNhe (5′-gat ggg ccc ttg gtg cta gct gag gagacg g-3′) (SEQ ID NO: 10) (98° C. for 1 second, 60° C. for 30 seconds,and 72° C. for 30 seconds), the amplified cDNA fragment of the heavychain was digested with SalI and NheI, and introduced into the N5KG1-ValLark vector (a modified vector of IDEC Pharmaceuticals, N5KG1 (U.S. Pat.No. 6,001,358)) that was cleaved with the same enzyme. The insertedsequence was confirmed to be identical to the one identified by directsequencing by identifying the sequence using the vector as a template.

The light chain was amplified by repeating 30 PCR cycles of 98° C. for 1second and 68° C. for 30 seconds using UMP (SMART RACE cDNAamplification Kit, Clontech) and the hk-2 primer (5′-GTT GAA GCT CTT TGTGAC GGG CGA GC-3′) (SEQ ID NO: 11). Further, 1 μL of this reactionsolution was used as a template, and 20 PCR cycles of 98° C. for 1second and 68° C. for 30 seconds were repeated using NUMP (SMART RACEcDNA amplification Kit, Clontech) and hk-6 (5′-TGGC GGG AAG ATG AAG ACAGAT GGT G-3′) (SEQ ID NO: 12). Thereafter, the amplified PCR product waspurified using a PCR purification kit (QIAGEN), and nucleotidesequencing was carried out using the hk-6 primer (SEQ ID NO: 12). Basedon the sequence information, the HD4 light chain specific primertnHD4Bgl (5′-ata tag atc tGC TGC TCA GTT AGG ACC CAG AGG GAA CC-3′) (SEQID NO: 13) and the HD8 light chain specific primer tnHD8Bgl (5′-ata tagatc tGG GAG TCA GAC CCA CTC AGG ACA CAG C-3′) (SEQ ID NO: 14) weresynthesized, and the resultant primer was used to determine the sequencealso from the opposite direction. PCR was carried out using a specificprimer and tnCkBsi (5′-aag aca gat ggt gca gcc acc gta cgt ttg at-3′)(SEQ ID NO: 15) (98° C. for 1 second, 60° C. for 30 seconds, and 72° C.for 30 seconds), the amplified cDNA fragment of the light chain wasdigested with BglII and BsiWI and introduced into the N5KG1-Val Larkvector that was cleaved with the same enzyme. The inserted sequence wasconfirmed to be identical to the one identified by direct sequencing byidentifying the sequence using the vector as a template.

DNAs encoding the variable region of the heavy chain, that of the lightchain of HD4, amino acid sequences of the variable region of the heavychain, and that of the light chain of HD4 are shown below.

<The Variable Region of the Heavy Chain of HD4> (SEQ ID NO: 16)

GTCGACCCAGCCCTGGGATTTTCAGGTGTTTTCAGGTGTTTTCATTTGGTGATCAGGACTGAACAGAGAGAACTCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAACTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTGGTGGTGATAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATCATGGTTCGGGGAGTTATTATCCCTACTGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCAGCTAGC<The Variable Region of the Heavy Chain of HD4> (SEQ ID NO: 17)

MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSGISGGGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGSGSYYPYWFDYWGQGTLVTVSSA<The Variable Region of the Light Chain of HD4> (SEQ ID NO: 18)

AGATCTGCTGCTCAGTTAGGACCCAGAGGGAACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAACTTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCCGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACG<The Variable Region of the Light Chain of HD4> (SEQ ID NO: 19)

METPAQLLFLLLLWLPDTTGELVLTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT

The translation initiation site in the heavy chain DNA is the ATG codonstarting from the 79th adenine (A) from the 5′-terminus of the sequenceas shown in SEQ ID NO: 16, and the boundary between the variable regionand the constant region of the antibody is located between the 504thadenine (A) and the 505th guanine (G) from the 5′-terminus. In the aminoacid sequence, the variable region of the heavy chain is a portionbetween the N-terminus and the 142nd serine (S) residue in the sequenceas shown in SEQ ID NO: 17, and a portion comprising the 143rd alanine(A) and thereafter is the constant region. The N-terminus of thepurified heavy chain protein was analyzed. This demonstrated that thesignal sequence of the heavy chain was a portion between the N-terminusand the 19th cysteine (C) of the sequence as shown in SEQ ID NO: 17, andthe N-terminus of the matured body was the 20th glutamic acid (E) of thesequence as shown in SEQ ID NO: 17. Accordingly, the matured portion inthe amino acid sequence as shown in SEQ ID NO: 17 is a portion betweenthe 20th glutamic acid and the 142nd serine.

The translation initiation site in the light chain DNA is the ATG codonstarting from the 35th A from the 5′-terminus of the sequence as shownin SEQ ID NO: 18, and the variable region is the portion between the5′-terminus and the 418th adenine (A). In the amino acid sequence, thevariable region is the portion between the N-terminus and the 128thlysine (K) of the sequence as shown in SEQ ID NO: 19. The N-terminus ofthe purified light chain protein was analyzed. This demonstrated thatthe signal sequence of the light chain was the portion between theN-terminus and the 20th glycine (G) of the sequence as shown in SEQ IDNO: 19, and the N-terminus of the matured body was the 21st glutamicacid (B) of the sequence as shown in SEQ ID NO: 19. Accordingly, thematured portion in the amino acid sequence as shown in SEQ ID NO: 19 isthe portion between the 21st glutamic acid and the 128th lysine.

DNAs encoding the variable region of the heavy chain, that of the lightchain of HD8, amino acid sequences of the variable region of the heavychain, and that of the light chain of HD8 are shown below.

<The Variable Region of the Heavy Chain of HD8> (SEQ ID NO: 20)

GTCGACTGGCTGACCAGGGCAGTCACCAGAGCTCCAGACAATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTTCAGCTGCAGCACTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACTGGATCAGGCAGTCCCCATCGAGGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAGTCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAAATTTCTATGGTTCGGAGACTTGTCATAAGAAGTATTACTGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTA GC<The Variable Region of the Heavy Chain of HD8> (SEQ ID NO: 21)

MSVSFLIFLPVLGLPWGVLSQVQLQHSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRIVINPDTSKNQFSLQLNSVTPEDTAVYYCARENFYGSETCHKKYYCYGMDVWGQGTTVTV SSAS<The Variable Region of the Light Chain of HD8> (SEQ ID NO: 22)

AGATCTGGGAGTCAGACCCACTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAACTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACG<The Variable Region of the Light Chain of HD8> (SEQ ID NO: 23)

MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTTTCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSFPLTFGGGTKVEIKRTV

The translation initiation site in the heavy chain DNA is the ATG codonstarting from the 41st adenine (A) from the 5′-terminus of the sequenceas shown in SEQ ID NO: 20, and the boundary between the variable regionand the constant region of the antibody is located between the 496thadenine (A) and the 497th guanine (G) from the 5′-terminus. In the aminoacid sequence, the variable region of the heavy chain is the portionbetween the N-terminus and the 152nd serine (S) residue in the sequenceas shown in SEQ ID NO: 21, and the portion comprising the 153rd alanine(A) and thereafter is the constant region. The N-terminus of thepurified heavy chain protein was analyzed. This demonstrated that thesignal sequence of the heavy chain was the portion between theN-terminus and the 20th serine (S) of the sequence as shown in SEQ IDNO: 21, and the N-terminus of the matured body was the 21st glutamine(Q) of the sequence as shown in SEQ ID NO: 21. Accordingly, the maturedportion in the amino acid sequence as shown in SEQ ID NO: 21 is theportion between the 21st glutamine and the 152nd serine.

The translation initiation site in the light chain DNA is the ATG codonstarting from the 34th A from the 5′-terminus of the sequence as shownin SEQ ID NO: 22, and the variable region is the portion between the5′-terminus and the 420th adenine (A). In the amino acid sequence, thevariable region is the portion between the N-terminus and the 129thlysine (K) of the sequence as shown in SEQ ID NO: 23. The N-terminus ofthe purified light chain protein was analyzed. This demonstrated thatthe signal sequence of the light chain is the portion between theN-terminus and the 22nd cysteine (C) of the sequence as shown in SEQ IDNO: 23, and the N-terminus of the matured body was the 23rd alanine (A)of the sequence as shown in SEQ ID NO: 23. Accordingly, the maturedportion in the amino acid sequence as shown in SEQ ID NO: 23 is theportion between the 23rd alanine and the 129th lysine.

TABLE 2 Nucleotide sequences of synthetic DNA No Primer Sequence (5′ to3′) Length SEQ ID NO. 1 hh-6 GGT CCG GGA GAT CAT GAG GGT GTC CCT 27 5 2hh-3 GTG CAC GCC GCT GGT CAG GGC GCC TG 26 6 3 hh-4 GGT GCC AGG GGG AAGACC GAT GG 23 7 4 tnHD4Sal ata tgt cga cCC AGC CCT GGG ATT TTC AGG TGTTTT C 37 8 5 tnHD8Sal ata tgt cga cTGG CTG ACC AGG GCA GTC ACC AGA G 359 6 tnCHNhe gat ggg ccc ttg gtg cta gct gag gag acg g 31 10 7 hk-2 GTTGAA GCT CTT TGT GAC GGG CGA GC 26 11 8 hk-6 T GGC GGG AAG ATG AAG ACAGAT GGT G 26 12 9 tnHD4Bgl ata tag atc tGC TGC TCA GTT AGG ACC CAG AGGGAA CC 38 13 10 tnHD8Bgl ata tag atc tGG GAG TCA GAC CCA CTC AGG ACA CAGC 37 14 11 tnCkBsi aag aca gat ggt gca gcc acc gta cgt ttg at 32 15

EXAMPLE 13 Preparation of Subclass Recombinant Vectors

The variable regions of the HD4 and HD8 antibodies were incorporatedinto the following variety of vectors. The N5KG1-Val Lark vector wasused for the IgG1 type, the N5KG4 Lark vector was used for the IgG4 type(both vectors were manufactured by IDEC Pharmaceuticals, modifiedvectors of N5KG1 were disclosed in U.S. Pat. No. 6,001,358, N5KG1-ValLark was modified while sustaining IgG1 as with N5KG1, and N5KG4 Larkwas rearranged to the IgG4 type), a vector in which the constant regionof the heavy chain of the N5KG1-Val Lark was rearranged into the IgG2type (N5KG2) was used for the IgG2 type, and the variable regions of HD4and HD8 were incorporated into the vector in the same manner as inExample 12. Separately, a vector in which the sequence CCC encodingproline (P) 331 according to the EU numbering system in the constantregion of the heavy chain that was incorporated into the IgG1 or IgG2vector was varied to TCC encoding serine (S) (Mi-Hua Tao et al., 1993,J. Exp. Med.) was prepared (hereafter referred to as N5KG1Ser andN5KG2Ser in that order), and the variable regions of HD4 and HD8 wereincorporated into the vector in the same manner as in Example 12. InFIG. 3 and Table 3 below, for example, the HD4 antibody having an IgG1subclass is referred to as HD4IgG1 or HD4G1, and the antibody thatexpresses the gene in which the sequence CCC encoding proline (P) 331according to the EU numbering system in the constant region of the heavychain was varied to TCC encoding serine (S) is referred to as HD4IgG1Seror HD4G1Ser.

TABLE 3 Names of recombinant vectors and produced recombinant antibodiesRecombinant antibody and its cytotoxic activity Anti- Recombinant ADCCCDC body Vector Subclass antibody activity activity HD4 N5KG1-Val LarkIgG1 HD4G1 + + HD4 N5KG2Ser IgG2Ser HD4G2Ser − − HD4 N5KG4 Lark IgG4HD4G4 − − HD8 N5KG1-Val Lark IgG1 HD8G1 + + HD8 N5KG1Ser IgG1SerHD8G1Ser + − HD8 N5KG2 IgG2 HD8G2 − + HD8 N5KG2Ser IgG2Ser HD8G2Ser − −HD8 N5KG4 Lark IgG4 HD8G4 − −

EXAMPLE 14 Preparation of Recombinant Antibody

The recombinant antibody-expressing vectors constructed in Examples 12and 13 were introduced into host cells to prepare a recombinantantibody-expressing cell. Examples of host cells for expression that canbe used include dhfr-deficient CHO cells (ATCC CRL-9096), CHO-Ras(Katakura Y., et al., Cytotechnology, 31: 103–109, 1999), and HEK293T(ATCC CRL-11268).

Vectors were introduced into host cells by electroporation, lipofection,or the like. Electroporation was carried out linearizing about 2 μg ofan antibody-expressing vector with a restriction enzyme, introducing thegenes into 4×10⁶CHO cells using Bio-Rad electrophoreter at 350 V and 500μF, and sowing them on a 96-well culture plate. Lipofection was carriedout using LipofectAMINE Plus (Gibco BRL) in accordance with theinstructions therefor. After introducing the vectors, an agent thatcorresponds to the selection marker for the expression vector was added,and the culture was continued. After the colonies were confirmed, theantibody-expressing cells were selected by the method described inExample 4. Antibodies were purified from the selected cells inaccordance with Example 8.

EXAMPLE 15 Examination of Cytotoxic Activity

Cytotoxic activities through an antibody were assayed. These activitiescomprised cytotoxic activity on the target cell in the presence of theNK cell or a cell with killer activity such as a neutrophil and anantibody, i.e., antibody-dependent cellular cytotoxicity (ADCC), andcytotoxic activity on the target cell in the presence of a complementand an antibody, i.e., complement-dependent cytotoxicity (CDC). Thehybridoma-derived HD4 and HD8 antibodies prepared in Example 8 andrecombinant antibodies derived from each of the CHO cells prepared inExample 14 were used. In this case, hIgG was used as a control.

In simple terms, radioactive chromium (Cr⁵¹) was incorporated into thecytoplasm of the target cell, and the amount of Cr⁵¹ released in theculture solution due to the cell death was measured based on the γ dose.

More specifically, 10⁶ Burkitt's lymphoma cells, Raji (ATCC CCL-86), asthe target cells were suspended in 15 μL of fetal calf serum (FCS), 50μL (37 MBq/mL) of Cr⁵¹-labeled sodium chromate (Perkin Elmer, hereafterreferred to as “Cr⁵¹”) was added, and culture was then conducted at 37°C. for 1 hour. Subsequently, 10 mL of medium was added, centrifugationwas carried out, and the medium was discarded. This procedure wasrepeated three times to remove Cr⁵¹ that was not incorporated in thecell.

In the ADCC assay, 200,000 mononuclear cells, which were derived fromthe peripheral blood of a healthy person obtained by the methoddescribed in Example 6, relative to 2,000 Cr⁵¹-labeled target cells(total volume: 200 μL) were cultured in a round-bottom, 96-well plate(Falcon) together with antibodies at each concentration level at 37° C.in the presence of 5% CO₂ for 4 hours.

In the CDC assay, human serum-derived complements (final concentration:5%, SIGMA) relative to 2,000 Cr⁵¹-labeled target cells (total volume:200 μL) were cultured in a round-bottom, 96-well plate together withantibodies at each concentration level at 37° C. in the presence of 5%CO₂ for 2 hours.

In both of the ADCC and CDC assays, the plate was subjected tocentrifugation after the culture in order to deposit cells. Thereafter,50 μL of supernatant was transferred to a powder scintillator-containing96-well plate (Lumaplate™-96, Packard) and dehydrated at 55° C. for 1.5hours. After confirming the dehydration, the plate was covered with adedicated-purpose cover (TopSeal™-A, 96-well Microplates, Packard), andthe γ dose was measured using a scintillation counter (TopCount,Packard).

The results are shown in FIG. 3 and Table 3. HD8IgG1Ser, HD8IgG2Ser,HD8IgG4, HD4G2Ser, and HD4G4 had no CDC activity, and HD81gG1, HD8IgG2,and HD4G1 had CDC activity. Only HD8IgG1, HD8IgG1Ser, and HD4G1 had ADCCactivity.

EXAMPLE 16 Effects of Recombinant Antibody on Tumor-bearing Mouse Model

Similar models as used in Example 11 were used to examine thepharmacological function of the HD8 recombinant antibody usingtumor-bearing mouse models.

At the outset, 5-week-old C.B-17/ICR-SCID mice (CLEA Japan, Inc.) werepurchased, anti-asialo GM1 antiserum (Wako Chemicals) was diluted, and10 μL each thereof was intravenously administered to each of the micewhen they were 6 weeks old. On the next day, Burkitt's lymphoma cellsRaji (ATCC CCL-86) were intravenously administered in amounts of 5×10⁶cells per mouse. Three days after the Raji transplantation, eachantibody was administered once in tail veins of mice in amounts of 0.1μg or 1 μg per mouse. The number of surviving mice after thetransplantation was observed.

The HD8G1Ser and HD8G2Ser prepared in Example 14 were administered oncein amounts of 0.1 or 1 μg per mouse. As an antibody negative control, agroup of mice to which the hIgG antibody used as a negative control inExample 9 was administered at 1 μg/head was provided. As a positivecontrol, a group of mice to which HD8G1 was administered at 1 μg/mousewas provided.

The results of the above experiment are shown in FIG. 4. All the samplesdied within 16 days after the Raji transplantation in the negativecontrol, i.e., the hIgG-administered group. The number of surviving mice20 days later is as follows. With the administration of 1 μg per mouse,all 6 samples survived except for the hIgG group (FIG. 4A), and with theadministration of 0.1 μg per mouse, all 6 samples in the HD8G2Ser groupand 2 samples in the HD8G1Ser group survived (FIG. 4B). The number ofsurviving mice 25 days later is as follows. With the administration of 1μg per mouse, 2 mice in the HD8G1Ser group, 1 mouse in the HD8G1 group,and 4 mice in the HD8G2Ser group survived (FIG. 4A), and with theadministration of 0.1 μg per mouse, 1 mouse only in the HD8G2Ser groupsurvived (FIG. 4B).

These results demonstrated that the HD8 recombinant antibodies, HD8G1Serand HD8G2Ser, also exhibited anti-tumor effects at very low dosages aswith HD8. Although HD8G2Ser had neither of ADCC or CDC activity, itexhibited beneficial effects on animal models.

EXAMPLE 17 Epitope Analyses of HD4, HD6, and HD8

The epitope of each antibody was analyzed by Western blotting inaccordance with a conventional technique. In simple terms, a celluloseor PVDF membrane was blocked with Block Ace (Yukijirushi), etc., eachantibody was allowed to react therewith at 1 μg/mL as a primary antibodyand at 0.5 μg/mL as a secondary antibody using HRP conjugated antirabbit IgG (DAKO), the HRP-labeled anti-human antibody (e.g., DAKO) wasallowed to react therewith, and the chemiluminescence was detected usinga chemiluminescent reagent (e.g., ECL Western blotting detectionreagent, Amersham Bioscience) and a chemiluminescence detector (e.g.,LAS-1000, Fuji Film).

(1) A membrane fraction was extracted from the HLA-DR-expressinglymphoma cell SKW 6.4 (ATCC TIB-215), and the HLA-DR protein waspurified from the anti-HLA-DR antibody (K28N, the name of produced cell:mouse-mouse hybridoma K28, deposited internationally at theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Feb. 22, 1994 under theaccession number of FERM BP4577) using an affinity column. The obtainedprotein was boiled using 4–20% gradient gel (Daiichi Pure Chemicals Co.,Ltd.) under nonreducing conditions (95° C., 5 minutes), electrophoresiswas carried out at a constant current of 25 mA for 1.5 hours per gel,and the resultant was transferred to a PVDF membrane at a constantcurrent of 150 mA for 1 hour per gel. Subsequently, Western blotanalysis was carried out in accordance with a conventional technique,and as a result, all of HD4, HD6, and HD8 were found to recognize theHLA-DR β chain located at approximately 30 Kda.

(2) Subsequently, regarding 199 amino acids in the extracellular regionof the HLA-DR β chain (DRB1*15011) (199 amino acids from amino acids 29to 227 in the amino acid sequence as shown in SEQ ID NO: 147 and thenucleotide sequence encoding the amino acid sequence as shown in SEQ IDNO: 147 is shown in SEQ ID NO: 146), 94 types of peptides in total (SEQID NOs: 52 to 145) were spot synthesized from the C terminus on thecellulose membrane by shifting two amino acids in the 13-mer peptides,and the N terminus was acetylated (JERINI Germany). The subsequentreaction was carried out based on conventional Western blot analysis(see, for example, Reineke, U. et al., (2001), “Epitope mapping withsynthetic peptides prepared by SPOT synthesis.” Antibody Engineering(Springer Lab Manual) Eds.: Kontermann/Dubel, 433–459). In the analysis,LumilmagerTM (Boehringer-Mannheim) was used to represent the colorintensity in each spot by numerical values.

The results are shown in FIG. 5. HD4 (FIG. 5A) exhibited potentreactivity with amino acids 61 to 73 and low reactivity with amino acids17 to 29, 63 to 75, and 65 to 77, and maximally bound to a peptidehaving the amino acid sequence as shown in SEQ ID NO: 82. HD6 (FIG. 5B)exhibited potent reactivity with amino acids 61 to 73 and low reactivitywith amino acids 57 to 69 and 59 to 71, and maximally bound to a peptidehaving the amino acid sequence as shown in SEQ ID NO: 82. HD8 (FIG. 5C)exhibited very potent reactivity with amino acids 61 to 73, 63 to 75,and 65 to 77 and potently bound to at least one peptide having the aminoacid sequences as shown in SEQ ID Nos: 82, 83, and 84. In contrast,based on the conformation of HLA-DR (see, for example, Dessen A et al.,Immunity (1997), 7, 473–481), amino acids 61 to 73 form the a helixstructure at the site where antigen-presenting peptides are retained.

(3) The β-chain polymorphisms of the 13 amino acids (amino acids 61 to73) that exhibit maximal reactivity with all of HD4, HD6, and HD8 weretaken into consideration, and 16 types of peptides includingsubstantially almost all of the currently known about 350 types ofpolymorphisms (see the IMGT/HLA database of EMBL-EBI, etc.) wereprepared. Further, 12 peptides (SEQ ID Nos: 40 to 51) in which eachamino acid has been substituted with alanine (guanidine if it isoriginally alanine) were subjected to Western blotting under similarconditions. In the analysis, LAS2000 and ImageGauge analyzing software(Fuji Photo Film) were used to represent the color intensity in eachspot by numerical values. FIG. 6 shows 28 types of peptide sequences andtheir reactivity with HD4, HD6, and HD8. The sequence to which eachantibody positively reacted was represented by + to ++++ depending onits intensity, and represented by − in case of negative reactivity.

Positive or negative reaction was judged in accordance with thefollowing criteria.

-   Less than 5% of the background: −-   5% to less than 10% of the background: +/−-   10% to less than 20% of the background: +-   20% to less than 30% of the background: ++-   30% to less than 50% of the background: +++-   50% or more of the background: ++++

HD8 positively reacted with all sequences except for those as shown inSEQ ID Nos: 48 to 51 in which amino acids 65, 66, 69, and 72 had beensubstituted with alanine. The amino acids 65, 66, 69, and 72 areconserved in substantially almost all of the discovered HLA-DR β chains(see the IMGT/HLA database of EMBL-EBI, etc.) Since they cover most ofthe HLA-DR β chain sequences including major sequences among those asshown in SEQ ID Nos: 24 to 39, HD8 is very highly likely to be aPan-HLA-DR antibody that binds to substantially almost all the HLA-DR βchains.

Furthermore, reactivity of HD8 was examined using the HLA-DR positivecell strain in the same manner as in Example 10. As a result, it wasfound to react with ARH77 (ATCC CRL-1621), Daudi (ATCC CCL-213),HS-Sultan (ATCC CRL-1484), IM-9 (ATCC CCL-159), MC/CAR (ATCC CRL-8083),Raji (ATCC CCL-86), Ramos (ATCC CRL-1596), RL (ATCC CRL-2261), SKW6.4(ATCC TIB-215), and the L-cell (ATCC CCL-1) in which DRB1*15011/DRA*0101were forcibly expressed and 5 specimens of human peripheral bloodmononuclear cells derived from a healthy Japanese person. It has not yetbeen discovered in a cell in which nonreactive HLA-DR is expressed.Further, it reacts with 15 out of 15 crab-eating monkey specimens and 1out of 1 chimpanzee specimen. In contrast, HD8 did not react with RPMI8226 (ATCC CCL-155), which is a B-cell strain in which HLA-DR is notexpressed.

INDUSTRIAL APPLICABILITY

The present invention provides a preventive or therapeutic agent fordiseases caused by HLA-DR-expressing cells, and more particularly, amolecule that is useful as a therapeutic agent for malignant tumors forpatients having substantially almost all HLA-DR polymorphisms.

Further, the present invention provides a immunosuppressive agent forsuppressing immunological activity associated with HLA-DR, and moreparticularly, a molecule that is useful as a therapeutic agent forrheumatisms.

All publications cited herein are incorporated herein by reference intheir entirety. A person skilled in the art would easily understand thatvarious modifications and changes are possible within the technical ideaand the scope of the invention as described in the attached claims. Thepresent invention is intended to include such modifications and changes.

1. An antibody or an antigen-binding fragment that binds to HLA-DR andthat is produced by the hybridoma HD8, deposited under accession number:FERM BP-7773.
 2. An antibody or an antigen-binding fragment that bindsto HLA-DR which comprises the heavy chain and light chain variableregions of an antibody produced by the hybridoma HD8, deposited underaccession number: FERM BP-7773.
 3. The antibody or an antigen-bindingfragment thereof according to claim 2, wherein the antibody subclass isIgG.
 4. The antibody or an antigen-binding fragment thereof according toclaim 3, wherein IgG is IgG1.
 5. The antibody or an antigen-bindingfragment thereof according to claim 4, wherein an amino acid sequence inthe constant region of the heavy chain is modified by substitution ofamino acid residue 331 according to the EU numbering system with serine.6. The antibody or an antigen-binding fragment thereof according toclaim 3, wherein IgG is IgG2.
 7. The antibody or an antigen-bindingfragment thereof according to claim 6, wherein an amino acid sequence inthe constant region of the heavy chain is modified by substitution ofamino acid residue 331 according to the EU numbering system with serine.8. The antibody or an antigen-binding fragment thereof according toclaim 3, wherein IgG is IgG3.
 9. The antibody or an antigen-bindingfragment thereof according to claim 3, wherein IgG is IgG4.
 10. Thehybridoma HD8, deposited under accession number FERM BP-7773.
 11. Anantibody or an antigen-binding fragment that binds to HLA-DR and thatcomprises (i) a first amino acid sequence, in its heavy chain,consisting of the sequence from the glutamine in amino acid position 21through the serine in amino acid position 152 of SEQ ID NO:21 and (ii) asecond amino acid sequence, in its light chain, consisting of thesequence from the alanine in amino acid position 23 through the lysinein amino acid position 129 of SEQ ID NO:23.
 12. The antibody or anantigen-binding fragment thereof according to claim 11, wherein theantibody subclass is IgG.
 13. The antibody or an antigen-bindingfragment thereof according to claim 12, wherein IgG is IgG1.
 14. Theantibody or an antigen-binding fragment thereof according to claim 13,wherein an amino acid sequence in the constant region of the heavy chainis modified by substitution of amino acid residue 331 according to theEU numbering system with serine.
 15. The antibody or an antigen-bindingfragment thereof according to claim 12, wherein IgG is IgG2.
 16. Theantibody or an antigen-binding fragment thereof according to claim 15,wherein an amino acid sequence in the constant region of the heavy chainis modified by substitution of amino acid residue 331 according to theEU numbering system with serine.
 17. The antibody or an antigen-bindingfragment thereof according to claim 12, wherein IgG is IgG3.
 18. Theantibody or an antigen-binding fragment thereof according to claim 12,wherein IgG is IgG4.
 19. An antibody or an antigen-binding fragment thatbinds to HLA-DR and that comprises (i) a first amino acid sequence, inits heavy chain, that is encoded by a nucleic acid sequence consistingof the sequence from the cytosine in nucleotide position 101 through theadenosine in nucleotide position 496 of SEQ ID NO:20 and (ii) a secondamino acid sequence, in its light chain, that is encoded by a nucleicacid sequence consisting of the sequence from the guanine in nucleotideposition 100 through the adenosine in amino acid position 420 of SEQ IDNO:22.
 20. The antibody HD8G1 Ser or an antigen-binding fragmentthereof, which is the antibody HD8 having an IgG1 subclass and aminoacid 331 in the constant region of the heavy chain according to the EUnumbering system being substituted with Ser.
 21. The antibody HD8G2 oran antigen-binding fragment thereof, which is the antibody HD8 having anIgG2 subclass and amino acid 331 in the constant region of the heavychain according to the EU numbering system being substituted with Ser.